Nucleoporins as drug targets for anti-proliferative therapeutics

ABSTRACT

Disclosed herein are methods of inhibiting nuclear pore complex assembly and inducing nuclear pore complex disassembly. Methods to screen for agents that inhibit nuclear pore assembly or induce nuclear pore complex disassembly are also disclosed.

CROSS-REFERENCE

This application is a U.S. national stage application of PCT/US18/41461,filed Jul. 10, 2018, and claims the benefit of U.S. ProvisionalApplication No. 62/531,257, filed Jul. 11, 2017, all of which areincorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION

Disclosed herein, in some embodiments, are methods of treating aproliferative disease or disorder in an individual in need thereof,comprising: administering to the individual a therapeutically effectiveamount of an agent that inhibits nuclear pore complex assembly, inducesnuclear pore complex disassembly, or inhibits nuclear pore complexfunction. In some instance, the agent inhibits expression of a componentof the nuclear pore complex. In some instances, the agent promotesdegradation of a component of the nuclear pore complex. In someinstances, the agent inhibits function of a component of the nuclearpore complex. In some instances, the agent modulates the expression of aregulator of the nuclear pore complex assembly. In some instances, theagent inhibits expression of a regulator of the nuclear pore complexassembly. In some instances, the agent inhibits expression of a positiveregulator of the nuclear pore complex assembly. In some instances, theagent induces expression of a regulator of the nuclear pore complexassembly. In some instances, the agent induces the expression ofnegative regulator of the nuclear pore complex assembly. In someinstances, the agent modulates the function of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsfunction of a regulator of the nuclear pore complex assembly. In someinstances, the agent inhibits function of a positive regulator of thenuclear pore complex assembly. In some instances, the agent inducesfunction of a regulator of the nuclear pore complex assembly. In someinstances, the agent induces the function of negative regulator of thenuclear pore complex assembly. In some instances, the agent modulatesthe expression of a regulator of the nuclear pore complex disassembly.In some instances, the agent induces expression of a regulator of thenuclear pore complex disassembly. In some instances, the agent inducesexpression of a positive regulator of the nuclear pore complexdisassembly. In some instances, the agent inhibits expression of aregulator of the nuclear pore complex disassembly. In some instances,the agent inhibits the expression of negative regulator of the nuclearpore complex disassembly. In some instances, the agent modulates thefunction of a regulator of the nuclear pore complex disassembly. In someinstances, the agent induces function of a regulator of the nuclear porecomplex disassembly. In some instances, the agent induces function of apositive regulator of the nuclear pore complex disassembly. In someinstances, the agent inhibits function of a regulator of the nuclearpore complex disassembly. In some instances, the agent inhibits thefunction of negative regulator of the nuclear pore complex disassembly.In some instances, the agent inhibits Nup35, Nup37, Nup43, Nup45, Nup50,Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits the expression of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88,Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188,Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits the function of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88,Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188,Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits the transport of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88,Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188,Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits the binding of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88,Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188,Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear pore complexto other components of the nuclear pore complex. In some instances, theagent inhibits Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155, Nup160,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex. Insome instances, the agent inhibits expression of Nup35, Nup37, Nup43,Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup93, Nup96,Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elys of thenuclear pore complex. In some instances, the agent inhibits function ofNup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85,Nup93, Nup96, Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1,Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elysof the nuclear pore complex. In some instances, the agent inhibitstransport of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155, Nup160,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex. Insome instances, the agent inhibits binding of Nup35, Nup37, Nup43,Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup93, Nup96,Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elys of thenuclear pore complex to other components of the nuclear pore complex. Insome instances, the agent is a small molecule. In some instances, theagent is a small interfering RNA (siRNA). In some instances, the agentis a short hairpin RNA (shRNA). In some instances, the agent is amicroRNA (miRNA). In some instances, the agent is a messenger RNA(mRNA). In some instances, the agent is a guideRNA (gRNA). In someinstances, the method further comprises administering an additionaltherapeutic agent. In some instances, the agent and the additionaltherapeutic agent are administered simultaneously. In some instances,the agent and the additional therapeutic agent are administeredsequentially. In some instances, the agent is administered beforeadministering the additional therapeutic agent. In some instances, theagent is administered after administering the additional therapeuticagent. In some instances, the disease or disorder is a neoplasticdisease. In some instances, the disease or disorder is caused by amalignant cell. In some instances, the disease or disorder is aninflammatory disease. In some instances, the disease or disorder iscancer. In some instances, the cancer has a mutation in the Ras gene. Insome instances, the mutation is a G12D mutation.

Disclosed herein, in some embodiments, is a pharmaceutical composition,comprising (a) an agent that inhibits nuclear pore complex assembly,induces nuclear pore complex disassembly, or inhibits nuclear porecomplex function; and (b) a pharmaceutically acceptable excipient. Insome instances, the agent inhibits expression of a component of thenuclear pore complex. In some instances, the agent promotes degradationof a component of the nuclear pore complex. In some instances, the agentinhibits function of a component of the nuclear pore complex. In someinstances, the agent modulates the expression of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsexpression of a regulator of the nuclear pore complex assembly. In someinstances, the agent inhibits expression of a positive regulator of thenuclear pore complex assembly. In some instances, the agent inducesexpression of a regulator of the nuclear pore complex assembly. In someinstances, the agent induces the expression of negative regulator of thenuclear pore complex assembly. In some instances, the agent modulatesthe function of a regulator of the nuclear pore complex assembly. Insome instances, the agent inhibits function of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsfunction of a positive regulator of the nuclear pore complex assembly.In some instances, the agent induces function of a regulator of thenuclear pore complex assembly. In some instances, the agent induces thefunction of negative regulator of the nuclear pore complex assembly. Insome instances, the agent modulates the expression of a regulator of thenuclear pore complex disassembly. In some instances, the agent inducesexpression of a regulator of the nuclear pore complex disassembly. Insome instances, the agent induces expression of a positive regulator ofthe nuclear pore complex disassembly. In some instances, the agentinhibits expression of a regulator of the nuclear pore complexdisassembly. In some instances, the agent inhibits the expression ofnegative regulator of the nuclear pore complex disassembly. In someinstances, the agent modulates the function of a regulator of thenuclear pore complex disassembly. In some instances, the agent inducesfunction of a regulator of the nuclear pore complex disassembly. In someinstances, the agent induces function of a positive regulator of thenuclear pore complex disassembly. In some instances, the agent inhibitsfunction of a regulator of the nuclear pore complex disassembly. In someinstances, the agent inhibits the function of negative regulator of thenuclear pore complex disassembly. In some instances, the agent inhibitsNup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85,Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160,Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121,Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of thenuclear pore complex. In some instances, the agent inhibits theexpression of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153,Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYSof the nuclear pore complex. In some instances, the agent inhibits thefunction of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153,Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYSof the nuclear pore complex. In some instances, the agent inhibits thetransport of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153,Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYSof the nuclear pore complex. In some instances, the agent inhibits thebinding of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153,Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYSof the nuclear pore complex to other components of the nuclear porecomplex. In some instances, the agent inhibits Nup35, Nup37, Nup43,Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup93, Nup96,Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elys of thenuclear pore complex. In some instances, the agent inhibits expressionof Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62,Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,or Elys of the nuclear pore complex. In some instances, the agentinhibits function of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155,Nup160, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex. Insome instances, the agent inhibits transport of Nup35, Nup37, Nup43,Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup93, Nup96,Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elys of thenuclear pore complex. In some instances, the agent inhibits binding ofNup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85,Nup93, Nup96, Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1,Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elysof the nuclear pore complex to other components of the nuclear porecomplex. In some instances, the agent is a small molecule. In someinstances, the agent is a small interfering RNA (siRNA). In someinstances, the agent is a short hairpin RNA (shRNA). In some instances,the agent is a microRNA (miRNA). In some instances, the agent is amessenger RNA (mRNA). In some instances, the agent is a guideRNA (gRNA).In some instances, the composition further comprises an additionaltherapeutic agent. In some instances, the additional therapeutic agentis an anti-proliferative agent. In some instances, the additionaltherapeutic agent is anti-cancer agent. In some instances, theadditional therapeutic agent is a chemotherapeutic agent. In someinstances, the additional therapeutic agent is a hormonal agent.

Disclosed herein, in some embodiments, are methods of screening for atherapeutic agent for treating a proliferative disease or disorder in anindividual in need thereof, the method comprising: (a) contacting a cellwith a test agent; (b) detecting (i) inhibition of nuclear pore complexassembly as compared to a control; or (ii) induction of nuclear porecomplex disassembly as compared to a control; and (c) identifying thetest agent as a therapeutic agent if the test agent reduces the numberof nuclear pore complexes in the cell as compared to the control. Insome instances, the cell is a tumor cell in vitro. In some instances,the cell is a tumor cell in vivo. In some instances, step (b) comprisesperforming: (i) cell proliferation or survival assay in vitro; and (ii)tumor development, growth or metastasis assay in vivo. In someinstances, the detection is by direct visualization. In some instances,the detection is by microscopy imaging, Western blot,immunohistochemistry, ELISA, SPARCL, fluorescent signal detector,chromatography, radioactive binding assay, a fluorescence binding assay,mass spectrometry, a kinetic exclusion assay, a crystallography assay,or live imaging. In some instances, the therapeutic agent is a smallmolecule. In some instances, the therapeutic agent is a smallinterfering RNA (siRNA). In some instances, the therapeutic agent is ashort hairpin RNA (shRNA). In some instances, the therapeutic agent is amicroRNA (miRNA). In some instances, the therapeutic agent is amessenger RNA (mRNA). In some instances, the therapeutic agent is aguideRNA (gRNA). In some instances, the therapeutic agent is anantisense oligonucleotide. In some instances, the therapeutic agent is apeptide. In some instances, the therapeutic agent is a peptidomimetic.In some instances, the therapeutic agent is an aptamer. In someinstances, the therapeutic agent targets a component of the nuclear porecomplex. In some instances, the therapeutic agent inhibits expression ofa component of the nuclear pore complex. In some instances, thetherapeutic agent promotes degradation of a component of the nuclearpore complex. In some instances, the therapeutic agent inhibits functionof a component of the nuclear pore complex. In some instances, thetherapeutic agent modulates the expression of a regulator of the nuclearpore complex assembly. In some instances, the therapeutic agent inhibitsexpression of a regulator of the nuclear pore complex assembly. In someinstances, the therapeutic agent inhibits expression of a positiveregulator of the nuclear pore complex assembly. In some instances, thetherapeutic agent induces expression of a regulator of the nuclear porecomplex assembly. In some instances, the therapeutic agent induces theexpression of negative regulator of the nuclear pore complex assembly.In some instances, the therapeutic agent modulates the function of aregulator of the nuclear pore complex assembly. In some instances, thetherapeutic agent inhibits function of a regulator of the nuclear porecomplex assembly. In some instances, the therapeutic agent inhibitsfunction of a positive regulator of the nuclear pore complex assembly.In some instances, the therapeutic agent induces function of a regulatorof the nuclear pore complex assembly. In some instances, the therapeuticagent induces the function of negative regulator of the nuclear porecomplex assembly. In some instances, the therapeutic agent modulates theexpression of a regulator of the nuclear pore complex disassembly. Insome instances, the therapeutic agent induces expression of a regulatorof the nuclear pore complex disassembly. In some instances, thetherapeutic agent induces expression of a positive regulator of thenuclear pore complex disassembly. In some instances, the therapeuticagent inhibits expression of a regulator of the nuclear pore complexdisassembly. In some instances, the therapeutic agent inhibits theexpression of negative regulator of the nuclear pore complexdisassembly. In some instances, the therapeutic agent modulates thefunction of a regulator of the nuclear pore complex disassembly. In someinstances, the therapeutic agent induces function of a regulator of thenuclear pore complex disassembly. In some instances, the therapeuticagent induces function of a positive regulator of the nuclear porecomplex disassembly. In some instances, the therapeutic agent inhibitsfunction of a regulator of the nuclear pore complex disassembly. In someinstances, the therapeutic agent inhibits the function of negativeregulator of the nuclear pore complex disassembly. In some instances,the therapeutic agent inhibits Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS of the nuclear pore complex. In some instances, thetherapeutic agent inhibits the expression of Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the therapeutic agent inhibits the functionof Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62,Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155,Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS ofthe nuclear pore complex. In some instances, the therapeutic agentinhibits the transport of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS of the nuclear pore complex. In some instances, thetherapeutic agent inhibits the binding of Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear pore complexto other components of the nuclear pore complex. In some instances, thetherapeutic agent inhibits Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155,Nup160, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex. Insome instances, the therapeutic agent inhibits expression of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup93,Nup96, Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elys of thenuclear pore complex. In some instances, the therapeutic agent inhibitsfunction of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155, Nup160,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex. Insome instances, the therapeutic agent inhibits transport of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup93,Nup96, Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elys of thenuclear pore complex. In some instances, the therapeutic agent inhibitsbinding of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155, Nup160,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex toother components of the nuclear pore complex. In some instances, thetherapeutic agent targets a component of the nuclear pore complexcomprising a protein binding/interacting pair, thereby disrupting thepair. In some instances, the protein binding/interacting pair isselected from a pair listed in Table 1. In some instances, the proteinbinding/interacting pair is selected from a pair listed in Table 2. Insome instances, the protein binding/interacting pair is selected from apair listed in Table 3. In some instances, the therapeutic agent targetsthe RNA or DNA of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS of the nuclear pore complex. In some instances, thetherapeutic agent binds to Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS of the nuclear pore complex.

Disclosed herein, in some embodiments, are methods of screening for atherapeutic agent for treating a proliferative disease or disorder in anindividual in need thereof, the method comprising: (a) contacting a cellwith a test agent; (b) detecting inhibition of nuclear transport ascompared to a control; and (c) identifying the test agent as atherapeutic agent if the test agent reduces the expression or activityof a component of nuclear pore complex in the cell as compared to thecontrol. In some instances, the cell is a tumor cell in vitro. In someinstances, the cell is a tumor cell in vivo. In some instances, step (b)comprises performing: (i) cell proliferation or survival assay in vitro;and (i) tumor development, growth or metastasis assay in vivo. In someinstances, the expression or activity of the component is measured bymicroscopy imaging, Western blot, immunohistochemistry, ELISA, SPARCL,fluorescent signal detector, chromatography, radioactive binding assay,a fluorescence binding assay, mass spectrometry, a kinetic exclusionassay, a crystallography assay, PCR, or gel electrophoresis. In someinstances, the therapeutic agent is a small molecule. In some instances,the therapeutic agent is a small interfering RNA (siRNA). In someinstances, the therapeutic agent is a short hairpin RNA (shRNA). In someinstances, the therapeutic agent is a microRNA (miRNA). In someinstances, the therapeutic agent is a messenger RNA (mRNA). In someinstances, the therapeutic agent is a guideRNA (gRNA). In someinstances, the therapeutic agent is an antisense oligonucleotide. Insome instances, the therapeutic agent is a peptide. In some instances,the therapeutic agent is a peptidomimetic. In some instances, thetherapeutic agent is an aptamer. In some instances, the therapeuticagent targets a component of the nuclear pore complex. In someinstances, the therapeutic agent inhibits expression of a component ofthe nuclear pore complex. In some instances, the therapeutic agentpromotes degradation of a component of the nuclear pore complex. In someinstances, the therapeutic agent inhibits function of a component of thenuclear pore complex. In some instances, the therapeutic agent modulatesthe expression of a regulator of the nuclear pore complex assembly. Insome instances, the therapeutic agent inhibits expression of a regulatorof the nuclear pore complex assembly. In some instances, the therapeuticagent inhibits expression of a positive regulator of the nuclear porecomplex assembly. In some instances, the therapeutic agent inducesexpression of a regulator of the nuclear pore complex assembly. In someinstances, the therapeutic agent induces the expression of negativeregulator of the nuclear pore complex assembly. In some instances, thetherapeutic agent modulates the function of a regulator of the nuclearpore complex assembly. In some instances, the therapeutic agent inhibitsfunction of a regulator of the nuclear pore complex assembly. In someinstances, the therapeutic agent inhibits function of a positiveregulator of the nuclear pore complex assembly. In some instances, thetherapeutic agent induces function of a regulator of the nuclear porecomplex assembly. In some instances, the therapeutic agent induces thefunction of negative regulator of the nuclear pore complex assembly. Insome instances, the therapeutic agent modulates the expression of aregulator of the nuclear pore complex disassembly. In some instances,the therapeutic agent induces expression of a regulator of the nuclearpore complex disassembly. In some instances, the therapeutic agentinduces expression of a positive regulator of the nuclear pore complexdisassembly. In some instances, the therapeutic agent inhibitsexpression of a regulator of the nuclear pore complex disassembly. Insome instances, the therapeutic agent inhibits the expression ofnegative regulator of the nuclear pore complex disassembly. In someinstances, the therapeutic agent modulates the function of a regulatorof the nuclear pore complex disassembly. In some instances, thetherapeutic agent induces function of a regulator of the nuclear porecomplex disassembly. In some instances, the therapeutic agent inducesfunction of a positive regulator of the nuclear pore complexdisassembly. In some instances, the therapeutic agent inhibits functionof a regulator of the nuclear pore complex disassembly. In someinstances, the therapeutic agent inhibits the function of negativeregulator of the nuclear pore complex disassembly. In some instances,the therapeutic agent inhibits Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS of the nuclear pore complex. In some instances, thetherapeutic agent inhibits the expression of Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the therapeutic agent inhibits the functionof Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62,Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155,Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS ofthe nuclear pore complex. In some instances, the therapeutic agentinhibits the transport of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS of the nuclear pore complex. In some instances, thetherapeutic agent inhibits the binding of Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear pore complexto other components of the nuclear pore complex. In some instances, thetherapeutic agent inhibits Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155,Nup160, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex. Insome instances, the therapeutic agent inhibits expression of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup93,Nup96, Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elys of thenuclear pore complex. In some instances, the therapeutic agent inhibitsfunction of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155, Nup160,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex. Insome instances, the therapeutic agent inhibits transport of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup93,Nup96, Nup107, Nup133, Nup155, Nup160, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, or Elys of thenuclear pore complex. In some instances, the therapeutic agent inhibitsbinding of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup93, Nup96, Nup107, Nup133, Nup155, Nup160,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, or Elys of the nuclear pore complex toother components of the nuclear pore complex. In some instances, thetherapeutic agent targets a component of the nuclear pore complexcomprising a protein binding/interacting pair, thereby disrupting thepair. In some instances, the protein binding/interacting pair isselected from a pair listed in Table 1. In some instances, the proteinbinding/interacting pair is selected from a pair listed in Table 2. Insome instances, the protein binding/interacting pair is selected from apair listed in Table 3. In some instances, the therapeutic agent targetsthe RNA or DNA of Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS of the nuclear pore complex. In some instances, thetherapeutic agent binds to Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS of the nuclear pore complex.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a model exemplifying how inhibition of nuclear pore complex(NPC) assembly affects the survival of cells with differentproliferating rates. In addition to having higher proliferation rates,transformed cells are addicted to the nuclear transport machinery whichmakes them even more sensitive to the inhibition of NPC assembly.

FIG. 2A exemplifies the depletion of essential NPC components (Nup160and NDC1) using specific shRNAs inhibits NPC assembly. FIG. 2Bexemplifies inhibition of NPC assembly with siRNA against Nup160inhibits cell proliferation and reduces cell number.

FIG. 3A-3B exemplifies that inhibition of NPC assembly with shRNAsagainst core nuclear pore complex components important for NPC assembly(assay 1: Nup160 and assay 2: NDC1) blocks the proliferation of cancercells (FIG. 3A), but does affect non-dividing/slow dividing cells (FIG.3B).

FIG. 4A exemplifies that inhibition of nuclear pore assembly depletesnuclear pores from proliferating myoblasts but not differentiated musclecells. FIG. 4B exemplifies that inhibition of nuclear pore assembly hasno or little effect on slow proliferating cells.

FIG. 5A exemplifies cell cycle analysis of A375 cells treated withControl or Nup160 shRNAs to inhibit nuclear pore complex assembly.Inhibition of NPC assembly increases the population of cells is subG1(dead/dying cells). FIG. 5B is an exemplary quantification of cells indifferent phases from FIG. 5A.

FIG. 6A exemplifies cell cycle analysis of Control, Nup160-depleted orNDC1-depleted A375 cells at different times after release from cellcycle synchronization. Inhibition of NPC assembly increases thepopulation of cells is subG1 (black bar) over time. SubG1 cellscorrespond to dying/dead cells. FIG. 6B is an exemplary quantificationof the % of SubG1 cells in Control, Nup160-depleted or NDC1-depleted atdifferent times after depletion.

FIG. 7A-7B exemplify the increase in apoptotic cells in A375 cellsdepleted of the nuclear pore complex Nup160 or NDC1, which are requiredfor pore assembly, when compared to the A375 Control cells when stainedwith the apoptotic marker Anexin V on day 6 after knockdown induction.FIG. 7C is an exemplary percentage of apoptotic cells in proliferatingcultures of Control, Nup160-depleted or NDC1-depleted cells quantifiedover time. FIG. 7D illustrates cell viability of Control,Nup160-depleted or NDC1-depleted cells quantified over time.

FIG. 8A-8B exemplifies the measurement of nuclear import rates in A375skin melanoma cancer cells using the nuclear transport reporterNLS—tomato—NES by fluorescence recovery after photobleaching (FRAP).A375 cells expressing control (SC) or Nup160 (N1) shRNAs weretransfected with the NLS—tomato—NES nuclear transport reporter andnuclear import was measured by Fluorescence recovery afterphotobleaching (FRAP). FIG. 8A exemplifies the recovery of the Tomatosignal inside the nucleus after photobleaching. FIG. 8B exemplifies aquantification of the fluorescent signal for the Tomato reporter 1 minafter photobleaching. Inhibition of nuclear pore complex assembly blocksnuclear import.

FIG. 9 exemplifies that inhibition of nuclear pore assembly inhibitscell proliferation and induces cell death in different cancer cells.

FIG. 10 is an exemplary quantification of the reduction in the signalfor NPCs at the surface of the nucleus when nuclear pore assembly isinhibited by Nup160 depletion.

FIG. 11 illustrates cells expressing and endogenous nuclear pore complexcomponent tagged with GFP using CRISPR technology imaged by confocalmicroscopy.

FIG. 12 illustrates tumor growth over time before or after the inductionof Nup160 shRNA (Day 14). Inhibition of nuclear pore assembly by Nup160depletion inhibits tumor growth and results in tumor remission.

FIG. 13 illustrates percentage change in tumor volume from the start tothe end of treatment (inhibition of nuclear pore assembly). Inhibitionof nuclear pore assembly inhibits tumor growth and results in tumorremission.

FIG. 14A-14B exemplify inhibition of nuclear pore assembly results insmaller tumors than controls when Control or Nup160-depleted tumors wereisolated at the end of treatment and their volumes were measured. FIG.14A shows a representative image of tumors carrying control or Nup160shRNAs, untreated or treated with doxycycline to induce shRNA andinhibition of nuclear pore assembly. FIG. 14B is an exemplaryquantification of tumor volume after treatment in doxycycline-treatedcontrol and Nup160 shRNA carrying tumors.

FIG. 15 exemplifies depletion of Nup160 in HT-29-induced tumors resultsin strong inhibition of nuclear pore complex assembly in vivo.

FIG. 16 illustrates tumor growth over time before or after the inductionof Nup160. Inhibition of nuclear pore assembly by Nup160 depletioninhibits tumor growth and results in tumor remission.

FIG. 17 illustrates percentage change in tumor volume from the start tothe end of treatment (inhibition of nuclear pore complex assembly).Inhibition of nuclear pore assembly inhibits tumor growth and results intumor remission.

FIG. 18A-18B exemplify inhibition of nuclear pore assembly results insmaller tumors than controls when Control or Nup160-depleted tumors wereisolated at the end of treatment and their weights measured. FIG. 18Ashows a representative image of tumors carrying control or Nup160shRNAs. Inhibition of nuclear pore assembly strongly inhibits tumorgrowth and results in tumor remission. FIG. 18B is an exemplaryquantification of tumor weight after treatment in doxycycline-treatedcontrol and Nup160 shRNA carrying tumors.

FIG. 19A-19B exemplify tumor growth in mice injected with B16F10 mousemelanoma cells expressing luciferase followed in vivo by opticalimaging. B16F10 mouse melanoma cells expressing luciferase were injectedsubcutaneously in NOD-SCID immunodeficient mice. Tumor development andgrowth was followed over time by live optical imaging with Xenogensystems (FIG. 19A). FIG. 19A exemplifies images of luciferase signal inlive mice at 1, 5 and 9 days after injection. Positive signal denotestumor formation. The luciferase signal over time in animals injectedwith B16F10 mouse melanoma cells was quantified (FIG. 19B). FIG. 19B isan exemplary quantification of luciferase signal at different dayspost-injection of B16F10 cells.

FIG. 20 illustrates B16F10 mouse melanoma cells injected in the liver ofwild type mice to generate liver-associated tumors. Tumors were allowedto grow and mice were injected with Control (SC) or Nup160 (N1) siRNAsmixed with the Invivofectamine siRNA delivery system for 2 weeks for invivo knockdown. Tumor size in liver and non-liver (metastasis) organswas determined by measuring tumor weight at the end of the experimentaltime.

FIG. 21A-21B exemplify inhibition of nuclear pore assembly has a lowereffect on normal cells compared to cancer cells. Melanoma cancer cells(A375) or normal fibroblasts (IMR90) were treated with shRNAs to inhibitnuclear pore assembly (shN1) and cell proliferation over time wascompared to control-treated cells (shSC). FIG. 21A exemplifies that A375grow faster than IMR90 and are more sensitive to nuclear pore complexinhibition. IMR90 cells numbers are not significantly affected byinhibition of nuclear pore assembly during the duration of treatmentwhich completely eliminates the A375 melanoma cells. FIG. 21B is anexemplary magnification of the vertical axes to depict the lower effectof nuclear pore complex assembly inhibition for normal cells.

FIG. 22A-22B exemplify cancer cells carrying Ras mutation (G12D) aremore sensitive to the inhibition of nuclear pore assembly. FIG. 22Aillustrates that cells carrying wild type (WT) or mutant Ras (MT) wheretreated with shRNAs to inhibit nuclear pore assembly (shN1) and cellproliferation over time was compared to control-treated cells (shSC).FIG. 22B illustrates the number of shN1-treated cells to control cellsdetermined over time and shown as percentage. Ras mutant cells show asignificant higher inhibition of cell proliferation than Ras wild typecells.

FIG. 23 exemplifies a detectable reduction in nuclear pore complexsignal when nuclear pore complex assembly is inhibited.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein, in some embodiments, are methods of treating aproliferative disease or disorder in an individual in need thereof,comprising: administering to the individual a therapeutically effectiveamount of an agent that inhibits nuclear pore complex assembly, inducesnuclear pore complex disassembly; or inhibits nuclear pore complexfunction.

Disclosed herein, in some embodiments, are also methods of screening fora therapeutic agent for treating a proliferative disease or disorder,the method comprising: (a) contacting a cell with a test agent; (b)detecting (i) inhibition of nuclear pore complex assembly as compared toa control; or (ii) induction of nuclear pore complex disassembly ascompared to a control; and (c) identifying the test agent as atherapeutic agent if the test agent reduces the number of nuclear porecomplexes in the cell as compared to the control. The methods disclosedherein, further comprises screening for a therapeutic agent for treatinga proliferative disease or disorder, the method comprising: (a)contacting a cell with a test agent; (b) detecting inhibition of nucleartransport as compared to a control; and (c) identifying the test agentas a therapeutic agent if the test agent reduces the expression, levels,or activity of a component of nuclear pore complex in the cell ascompared to the control.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof any subject matter claimed. In this application, the use of thesingular includes the plural unless specifically stated otherwise. Itmust be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. In this application, theuse of “or” means “and/or” unless stated otherwise. Furthermore, use ofthe term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error, e.g., ±5%, ±10%, or ±15%.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)”are used interchangeably and mean any mammal. In some embodiments, themammal is a human. In some embodiments, the mammal is a non-human. Noneof the terms require or are limited to situations characterized by thesupervision (e.g. constant or intermittent) of a health care worker(e.g. a doctor, a registered nurse, a nurse practitioner, a physician'sassistant, an orderly or a hospice worker).

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology or symptoms of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. For example the term “treat” or “treating”with respect to a proliferative disorder refers to stopping theprogression of said disorder, slowing down, or amelioration of symptomsassociated with the presence of said cells, causing apoptosis of cellscausing the disorder. Treatment of an individual suffering from aproliferative disease or disorder refers to a decrease and eliminationof the cells causing the disease in an individual. For example, adecrease or elimination of tumor cells in an individual suffering fromcancer.

By “therapeutically effective amount” is meant an amount of a compounddescribed herein effective to yield the desired therapeutic response.For example, an amount effective to inhibit nuclear pore complexassembly, induce nuclear pore complex disassembly or inhibit nuclearpore complex function, thereby targeting the cells causing the disorder.The therapeutically effective amount will vary with such factors as theparticular condition being treated, the physical condition of thepatient, the type of mammal or animal being treated, the duration of thetreatment, the nature of concurrent therapy (if any), and the specificformulations employed and the structure of the agents or itsderivatives.

Nuclear Pore Complex

A hallmark of human cells is that the chromosomes (DNA) are housedinside a membrane structure known as the cell nucleus. The nucleus hasall the genetic material and is considered the control center of thecell. In order to regulate the activity of genes in the DNA inside thenucleus, cells need to be able to move molecules in and out of thiscompartment. Nuclear pore complexes (NPCs) are large protein channelsthat act as the doors of the nucleus. NPCs are multiprotein complexesbuilt from 33 different proteins known as nucleoporins. These channelsrepresent the gateway into the nuclear compartment and control theentrance and exit of all molecules in a highly regulated and efficientmanner. Since numerous proteins, including oncogenes and tumorsuppressors, need to access the genome to perform their functions,alterations in the nuclear transport machinery are observed in cancercells. No inhibitors of the nuclear pore complexes have been developedor tested as antineoplastic or antiproliferative agents. In someinstances, targeting the activity of the nuclear transport machineryaffects the survival of cancer cells. In some instances, targeting theactivity of the nuclear transport machinery affects the survival ofexcessively proliferative cells.

Inhibition of nuclear pore complex assembly, instead of thenucleocytoplasmic transport process, is beneficial for the treatment ofdisorders, such as neoplastic and proliferative disorders, for severalreasons. First, differently from nucleocytoplasmic transport, nuclearpore complex assembly is restricted to proliferating cells. Thus,blocking this process does not affect non-dividing cells, such asneurons, or muscle, which minimizes its effect on normal tissues andreduces undesired toxicity. Second, since inhibition of nuclear porecomplex assembly only occurs during cell division, cells that dividemore, faster or are more addicted to nuclear transport, such asmalignant cells, are specifically and more strongly affected. Third, incancer and other proliferative diseases, malignant cells showupregulated cell division either by misregulation of cell proliferationfactors, the inhibition of cell death pathways, or both. To controlthese processes, cells still need to regulate the activity of genesinside the nucleus. Elimination of the transport channels that allow themovement of molecules in and out of the nucleus is incompatible with thefunction of these cells and affects abnormally proliferating cellsindependently of the processes or pathways that they have mutated.Therefore, a therapy based on nuclear pore complex assembly inhibitionis effective in a widespread of proliferative and neoplastic diseasesindependently of their causes. Fourth, many current therapies forneoplastic or other proliferative disorders rely on inhibiting differentsignaling pathways, either at the level of surface receptors (such asEGFR inhibitors), or downstream effectors of the signaling cascades(e.g. ERK and ALK inhibitors). An important and very common problem thatthese therapies face is the rapid appearance of resistant cells, whichloose the sensitivity to the inhibitors. In many cases, cells overcomethe effect of the inhibitors by switching to, or over-activating, adifferent signaling pathway. But because all signaling pathways resultin the activation/repression of genes inside the nucleus and, thus,necessarily require molecules to enter and exit nucleus, eliminating thedoors to the nucleus (namely nuclear pore complexes) by inhibiting theirassembly prevents the cells on using other signaling cascades toovercome inhibition. Therefore, inhibiting nuclear pore complex assemblyhas a strong effect on the survival of highly proliferating cells withsignificantly lower chances of resistance than conventional approachesbased on the inhibition of cell signaling. In some embodiments, thepresent disclosure exemplifies that inhibiting the assembly of nuclearpore complexes: 1) reduces proliferation and induces death in cancercells without significantly affecting cells that do not divide or divideless frequently; 2) inhibits tumor growth; 3) induces cancer cell deathwithin the tumor; and 4) results in tumor remission and strongly reducestumor size in animals.

In the same way as nuclear pore complex assembly, disassembly of thisstructure only takes place in dividing cells. Stimulating the process ofnuclear pore complex disassembly in dividing cells also results in thesame reduction in nuclear pore complexes and affects the proliferationand survival of malignant cells as disclosed above. Identifyingregulators of nuclear pore complex disassembly is also a promisingstrategy for the treatment of proliferative diseases. In some instances,inhibiting nuclear pore complex assembly, inducing nuclear pore complexdisassembly, and inhibiting nuclear pore complex function by targetingdifferent nuclear pore complex components triggers cell death ofexcessively proliferating cells. In some instances, inhibiting nuclearpore complex assembly, inducing nuclear pore complex disassembly, andinhibiting nuclear pore complex function by targeting different nuclearpore complex components triggers cell death in cancer cells. In someinstances, inhibiting nuclear pore complex assembly, inducing nuclearpore complex disassembly, and inhibiting nuclear pore complex functionby targeting different nuclear pore complex components triggers celldeath of excessively proliferating cells in proliferative disease ordisorder. In some instances, the proliferative disease is cancer. Insome instances, the proliferative disease is atherosclerosis. In someinstances, the proliferative disease is various forms of arthritis. Insome instances, the proliferative disease is rheumatoid arthritis. Insome instances, the proliferative disease is psoriasis. In someinstances, the proliferative disease is various forms of fibrosis. Insome instances, the proliferative disease is idiopathic pulmonaryfibrosis. In some instances, the proliferative disease is scleroderma.In some instances, the proliferative disease is cirrhosis of the liver.In some instances, the proliferative disease is benign prostatichyperplasia. In some instances, the proliferative disease is abnormalscar formation. In some instances, the proliferative disease isinflammatory bowel disease.

Nucleoporins

Nucleoporins are a family of proteins that are the constituent buildingblocks of the nuclear pore complex. Nucleoporins mediate transport ofmacromolecules between the cell nucleus and cytoplasm in eukaryotes. Insome instances, certain nucleoporins form the structural scaffolding ofthe nuclear pore complex. In some instances, certain nucleoporinsfunction by interacting with transport molecules known as karyopherins.There are three types of nucleoporins, namely structural nucleoporins,membrane nucleoporins, and FG-nucleoporins. Structural nucleoporins formthe ring portion of the nuclear pore complex. They span the membrane ofthe nuclear envelope and are referred to as the scaffolding of thenuclear pore. Two sub-complexes of structural nucleoporins form the twoscaffold rings of each nuclear pore. Membrane nucleoporins are localizedto the curvature of a nuclear pore. These proteins are embedded withinthe nuclear membrane at the region where the inner and outer leafletsconnect. FG-nucleoporins are so named because they contain repeats ofthe amino acid residues phenylalanine and glycine. FG-repeats are smallhydrophobic segments that break up long stretches of hydrophilic aminoacids. These FG-repeat segments are found in long random-coil portionsof the protein which stretch into the channel of nuclear pores and areprimarily responsible for the selective permeability of nuclear porecomplexes. These segments of FG-nucleoporins form a mass of chains whichallow smaller molecules to diffuse through, but exclude largehydrophilic macromolecules. These large molecules are only able to crossa nuclear pore if they are accompanied by a transport receptor moleculethat temporarily interacts with a nucleoporin's FG-repeat segment.FG-nucleoporins also contain a globular portion that serves as an anchorfor attachment to the nuclear pore complex.

The family of nucleoporins comprises of Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS. Nucleoporins have beenshown to form various sub-complexes with one another. The most common ofthese complexes or binding/interacting pairs are listed in Table 1.

TABLE 1 Nucleoporin protein binding or interacting pairs Nup133-Nup160Nup358(RanBP2)-Nup188 Nup35- Nup358(RanBP2) Nup133-Nup107Nup358(RanBP2)-Nup93 Nup50-Nup98 Nup133-Nup37 Nup358(RanBP2)-Nup160Nup50-Nup153 Nup133-Nup43 Nup358(RanBP2)-Seh1L Nup153-Nup54 Nup133-Nup50Nup358(RanBP2)-Sec13 Nup153-Nup62 Nup133-Nup153 Nup93-Nup35 Nup153-Nup88Nup133-Nup155 Nup93-Nup62 Nup153-Nup93 Nup133-Nup214 Nup93-Nup54Nup153-Nup98 Nup133-Nup98 Nup93-Nup58 Nup153-Nup155 Nup133-Sec13Nup93-Nup45 Nup153-Nup205 Nup133-Seh1L Nup93-Nup98 Nup153-Nup214Nup107-Nup96 Nup93-Nup205 Nup153-hCG1(Nup12) Nup107-Nup50 Nup93-Nup214ELYS-Nup37 Nup107-Nup54 Nup205-Nup155 ELYS -Nup43 Nup107-Nup62Nup205-Nup35 ELYS -Nup75(Nup85) Nup107-Nup88 Nup205-Nup98 ELYS -Nup96Nup107-Nup93 Nup205-Nup188 ELYS -Nup107 Nup107-Nup98 Nup205-Nup214 ELYS-Nup133 Nup107-Nup153 Nup205-Nup358 ELYS -Nup160 Nup107-Nup155 Nup205-hCG1(Nupl2) ELYS -Sec13 Nup107-Nup205 Nup155-Nup35 ELYS -Sehl1Nup107-Nup214 Nup155-Nup37 ELYS-Nup35 Nup107-Sec13 Nup155-Nup62ELYS-Nup98 Nup107-Seh1L Nup155-Nup54 Nup62-Nup54 Nup96-Sec13 Nup155-Nup58 Nup62-Nup88 Nup96-Nup75(Nup85) Nup155- Nup45 Nup62-Nup98Nup96-Seh1L Nup155-Nup98 Nup62-Nup188 Nup96- Nup160 Nup155-Nup214Nup62-Nup214 Nup96-Nup37 Nup98-Nup88 Nup62-Nup358(RanBP2) Nup96-Nup155Pom121-NDC1 Nup98-Nup54 Nup160-Nup50 Pom121-Nup35 Nup98-Nup214Nup160-Nup37 Pom121-Nup43 Nup98-Nup358(RanBP2) Nup160-Nup43Pom121-Nup75(Nup85) TPR-Nup93 Nup160-Nup107 Pom121-Nup133 TPR-Nup98Nup160-SehL1 Pom121-Nup153 TPR-Nup107 Nup160-Sec13 Pom121-Nup160TPR-Nup153 Nup160-Nup153 Pom121-Pom121 TPR-Nup205 Nup160-Nup155 NDC1-NDC1 RAE1-Nup54 Nup160-Nup214 NDC1-Nup37 RAE1-Nup62 Nup160-Nup98NDC1-Nup43 RAE1-Nup88 Nup37-Sehl1 NDC1-Nup75(Nup85) RAE1-Nup93Nup37-Sec13 NDC1-Nup96 RAE1-Nup98 Nup37-Nup358(RanBP2) NDC1-Nup107RAE1-Nup107 Nup37-Nup43 NDC1-Nup133 RAE1-Nup153 Nup37-Nup75(Nup85)NDC1-Nup160 RAE1-Nup188 Nup37-Nup98 NDC1-Sec13 RAE1-Nup214 Nup37-Nup107NDC1-Sehl1 RAE1-Nup358(RanBP2) Nup37-Nup153 NDC1-ELYS RAE1-hCG1(Nupl2)Nup43-Sehl1 NDC1-Nup35 GLE1-Nup35 Nup43-Sec13 NDC1-Nup93 GLE1-Nup98Nup43-Nup50 NDC1-Nup98 GLE1-Nup107 Nup43-Nup358(RanBP2) NDC1-Nup153GLE1-Nup153 Nup43-Nup75(Nup85) NDC1-Nup155 GLE1-Nup155 Nup43-Nup98NDC1-Nup188 GLE1-NupSeh1 Nup43-Nup107 NDC1-Nup205 GLE1-Nup358(RanBP2)Nup43-Nup153 NDC1-Nup58 GLE1-Nup58 Nup43-Nup155 NDC1-Nup358(RanBP2)GLE1-ALADIN Nup43-Nup214 Nup58-Nup35 ALADIN-NDC 1 Nup75(Nup85)-Seh1LNup58-Nup54 ALADIN-Nup35 Nup75(Nup85)-Sec13 Nup58-Nup62 ALADIN-Nup98Nup75(Nup85)-Nup50 Nup58-Nup98 ALADIN-Nup107 Nup75(Nup85)-Nup62Nup58-Nup107 ALADIN-Nup153 Nup75(Nup85)-Nup88 Nup58-Nup188 ALADIN-Nup358Nup75(Nup85)-Nup98 Nup58-Nup205 ALADIN-Seh1L Nup75(Nup85)-Nup107Nup58-Nup214 Nup210-Nup98 Nup75(Nup85)-Nup153 Nup58-hCG1(Nup12)Nup210-Nup107 Nup75(Nup85)-Nup155 Nup58-Nup358(RanBP2) Nup210-Nup153Nup75(Nup85)-Nup160 hCG1(Nupl2)-Nup35 Nup210-Nup214 Nup75(Nup85)-Nup214hCG1(Nupl2)-Nup98 Nup210-Seh1L Sec13-Seh1 hCG1(Nupl2)-Nup107 Nup210-NDC1Sec13-Nup98 hCG1(Nupl2)-Nup214 Nup210-Pom121 Sec13-Nup155hCG1(Nupl2)-Nup358(RanBP2) Nup210-Nup210 Seh1-Nup88 Nup188-Nup35Nup210-Nup37 Seh1-Nup93 Nup188-Nup93 Nup210-Nup43 Seh1-Nup98Nup188-Nup98 Nup210-Nup75(Nup85) Seh1-Nup153 Nup188-Nup107 Nup210-Nup96Seh1-Nup155 Nup188-Nup153 Nup210-Nup107 Seh1-Nup205 Nup188-Nup214Nup210-Nup133 Seh1-Nup214 Nup188-Seh1L Nup210-Nup160 Seh1-Rae1Nup188-hCG1(Nup12) Nup210-Nup35 Nup214-Nup88 Nup35-Nup50 Nup210-Nup93Nup214-Nup358(RanBP2) Nup35-Nup54 Nup210-Nup155 Nup88-Nup358(RanBP2)Nup35-Nup62 Nup210-Nup188 Nup358(RanBP2)-Nup50 Nup35-Nup88 Nup210-Nup205Nup358(RanBP2)-Nup75(Nup85) Nup35-Nup98 Nup210-Seh1LNup358(RanBP2)-Nup107 Nup35-Nup107 Nup210-Sec13 Nup358(RanBP2)-Nup133Nup35-Nup153 Nup210-ELYS Nup358(RanBP2)-Nup153 Nup35-Nup214 NDC1-Nup45Nup358(RanBP2)-Nup155 Nup35-Seh1L GLE1-Nup45 Nup45-Nup35 Nup133-Nup96Pom121-Sec13 Nup45-Nup54 Pom133-Nup75(Nup85) Pom121-ELYS Nup45-Nup62Nup133-Nup35 Pom121-Nup93 Nup45-Nup98 Nup133-Nup93 Pom121-Nup155Nup45-Nup107 Nup133-Nup188 Pom121-Nup188 Nup45-Nup188 Nup133-Nup205Pom121-Nup205 Nup45-Nup205 Nup107-Pom121 Nup43-Nup96 Nup45-Nup214Pom121-Nup96 Nup43-Nup35 Nup45-hCG1(Nupl2) Pom121-Nup96 Nup43-Nup93Nup45-Nup358(RanBP2) Pom121-Seh1 Nup43-Nup188 Nup43-Nup205 Nup188-Nup62Nup96-Nup205 Nup93-Nup155 Nup62-Nup62 Nup160-Nup35 Nup205-Nup54Nup96-Nup35 Nup160-Nup93 Nup205-Nup62 Nup96-Nup93 Nup160-Nup188Nup188-Nup54 Nup96-Nup188 Nup160-Nup205 Sec13-Nup35 Nup75(Nup85)-Nup35Nup37-Nup35 Sec13-Nup93 Nup75(Nup85)-Nup93 Nup37-Nup93 Sec13-Nup188Nup75(Nup85)-Nup188 Nup37-Nup188 Sec13-Nup205 Nup75(Nup85)-Nup205Nup45-Nup45 Nup155-Nup188 Nup37-205 Nup45-Nup58 Nup54-Nup54 Nup54-Nup58Nup58-Nup58

TABLE 2 Nucleoporin protein interacting pair candidates to disrupt NPCassembly Nup133-Nup160 Nup93-Nup35 ELYS-Nup37 Nup133-Nup107 Nup93-Nup205ELYS -Nup43 Nup133-Nup37 Nup205-Nup155 ELYS -Nup75 (Nup85) Nup133-Nup43Nup205-Nup35 ELYS -Nup96 Nup133-Nup155 Nup205-Nup188 ELYS -Nup160Nup133-Sec13 Nup205-Nup214 ELYS -Sec13 Nup133-Seh1L Nup155-Nup35 ELYS-Sehl1 Nup133-Nup96 Nup155-Nup37 ELYS-Nup35 Pom133-Nup75(Nup85)Pom121-NDC1 ELYS-Nup98 Nup133-Nup35 Pom121-Nup35 Nup160-Nup37Nup133-Nup93 Pom121-Nup43 Nup160-Nup43 Nup133-Nup188 Pom121-Nup75Nup160-SehL1 (Nup85) Nup133-Nup205 Pom121-Nup160 Nup160-Sec13Pom121-Nup133 Pom121-Pom121 Nup160-Nup155 ELYS -Nup133 Pom121-Nup96Pom121-Nup205 NDC1-Nup133 Pom121-Nup96 Nup37-205 Nup107-Nup205Pom121-Seh1 Nup37-Nup35 Nup107-Sec13 Pom121-Sec13 Nup37-Nup93Nup107-Seh1L Pom121-ELYS Nup37-Nup188 Nup107-Nup96 Pom121-Nup93Nup37-Sehl1 Nup107-Nup93 Pom121-Nup155 Nup37-Sec13 Nup37-Nup107Pom121-Nup188 Nup37-Nup43 ELYS -Nup107 NDC1-Nup160 Nup37-Nup75 (Nup85)NDC1-Nup107 NDC1-Sec13 Nup43-Sehl1 Nup160-Nup107 NDC1-Sehl1 Nup43-Sec13Nup43-Nup107 NDC1-ELYS Nup43-Nup75 (Nup85) Nup35-Nup107 NDC1-Nup35Nup43-Nup155 Nup188-Nup107 NDC1-Nup93 Nup43-Nup96 Nup75(Nup85)-Nup107NDC1-Nup155 Nup43-Nup35 Nup107-Nup155 NDC1-Nup188 Nup43-Nup93Nup107-Pom121 NDC1-Nup205 Nup43-Nup188 Sec13-Seh1 NDC1-NDC1 Nup43-Nup205Sec13-Nup155 NDC1-Nup37 Nup188-Nup35 Seh1-Nup93 NDC1-Nup43 Nup188-Nup93Seh1-Nup155 NDC1-Nup75 Nup188-Seh1L (Nup85) Seh1-Nup205 NDC1-Nup96Nup35-Seh1L Nup75(Nup85)-Seh1L Sec13-Nup35 Nup160-Nup35Nup75(Nup85)-Sec13 Sec13-Nup93 Nup160-Nup93 Nup75(Nup85)-Nup155Sec13-Nup188 Nup160-Nup188 Nup75(Nup85)-Nup160 Sec13-Nup205Nup160-Nup205 Nup96-Nup75(Nup85) Nup93-Nup155 Nup96-Sec13Nup75(Nup85)-Nup35 Nup155-Nup188 Nup96-Seh1L Nup75(Nup85)-Nup93 Nup96-Nup160 Nup96-Nup35 Nup75(Nup85)-Nup188 Nup96-Nup37 Nup96-Nup93Nup75(Nup85)-Nup205 Nup96-Nup155 Nup96-Nup188 Nup96-Nup205

TABLE 3 Nucleoporin protein interacting pair candidates to disrupt NPCassembly or function Nup35-Nup45 Nup45-Nup45 Nup62-Nup45 Nup35-Nup54Nup45-Nup54 Nup62-Nup54 Nup35-Nup58 Nup45-Nup58 Nup62-Nup58 Nup35-Nup62Nup54-Nup54 Nup62-Nup62 Nup93-Nup45 Nup54-Nup58 Nup205-Nup45 Nup93-Nup54Nup58-Nup58 Nup205-Nup54 Nup93-Nup58 Nup188-Nup45 Nup205-Nup58Nup93-Nup62 Nup188-Nup54 Nup205-Nup62 Nup155-Nup45 Nup188-Nup58Nup155-Nup58 Nup155-Nup54 Nup188-Nup62 Nup155-Nup62

Disclosed herein, in some embodiments, is a method for treating aproliferative disease or disorder in an individual in need thereof,comprising: administering to the individual a therapeutically effectiveamount of an agent that inhibits nuclear pore complex assembly, inducesnuclear pore complex disassembly, or inhibits nuclear pore complexfunction. A pharmaceutical composition comprising an effective amount ofsuch an agent of interest and a pharmaceutically acceptable excipient isalso encompassed by the disclosure.

Exemplary Agents

Disclosed herein, in some embodiments, are agents that inhibit nuclearpore complex assembly, induce nuclear pore complex disassembly, orinhibit nuclear pore complex function. In some instances, the agent is asmall molecule. In some instances, the agent is a small interfering RNA(siRNA). In some instances, the agent is a short hairpin RNA (shRNA). Insome instances, the agent is a microRNA (miRNA). In some instances, theagent is a messenger RNA (mRNA). In some instances, the agent is aguideRNA (gRNA). In some instances, the agent is an antisenseoligonucleotide. In some instances, the agent is a peptide. In someinstances, the agent is a peptidomimetic. In some instances, the agentis an aptamer.

Small Molecule

Small molecule is a molecule that binds to a specific biologicaltarget—such as a specific protein or nucleic acid—and acts as aneffector, altering the activity or function of the target. In someembodiments, disclosed herein, the methods comprise an agent thatinhibits nuclear pore complex assembly, induces nuclear pore complexdisassembly, or inhibits nuclear pore complex function. In someinstances, the agent is a small molecule. In some instances, the smallmolecule binds to the nucleic acid or protein including, but not limitedto, Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62,Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155,Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS ofthe nuclear pore complex. In some instances, the small molecule disruptsa protein binding/interacting pair comprising pairs listed in Table 1.In some instances, the small molecule disrupts a proteinbinding/interacting pair comprising pairs listed in Table 2. In someinstances, the small molecule disrupts a protein binding/interactingpair comprising pairs listed in Table 3. In some instances, the smallmolecule reduces synthesis of a protein, thereby inhibiting nuclear porecomplex assembly, inducing nuclear pore complex disassembly, orinhibiting nuclear pore complex function.

Small Interfering RNA (siRNA)

Small interfering RNA (siRNA), sometimes known as short interfering RNAor silencing RNA, is a class of double-stranded RNA molecules, 20-25base pairs in length, similar to miRNA, and operates within the RNAinterference (RNAi) pathway. It interferes with the expression ofspecific genes with complementary nucleotide sequences by degrading mRNAafter transcription, resulting in no translation. In some embodiments,disclosed herein, the methods comprise an agent that inhibits nuclearpore complex assembly, induces nuclear pore complex disassembly, orinhibits nuclear pore complex function. In some instances, the agent isa small interfering RNA (siRNA). In some instances, the siRNA has asequence complementary to the target sequence of a nuclear pore complexcomponent including, but not limited to, Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS. In some instances, thesiRNA reduces synthesis of a protein, thereby inhibiting nuclear porecomplex assembly, inducing nuclear pore complex disassembly, orinhibiting nuclear pore complex function.

Short Hairpin RNA (shRNA)

Short hairpin RNA (shRNA) or small hairpin RNA (shRNA/Hairpin Vector) isan artificial RNA molecule with a tight hairpin turn that is used tosilence target gene expression via RNA interference (RNAi). Expressionof shRNA in cells is typically accomplished by delivery of plasmids orthrough viral or bacterial vectors. Once the vector has integrated intothe host genome, the shRNA is then transcribed in the nucleus and theproduct mimics pri-microRNA (pri-miRNA) and is processed by Drosha. Theresulting pre-shRNA is exported from the nucleus and processed by Dicerand loaded into the RNA-induced silencing complex (RISC). The sense(passenger) strand is degraded. The antisense (guide) strand directsRISC to mRNA that has a complementary sequence. In the case of perfectcomplementarity, RISC cleaves the mRNA. In the case of imperfectcomplementarity, RISC represses translation of the mRNA. In both ofthese cases, the shRNA leads to target gene silencing. In someembodiments, disclosed herein, the methods comprise an agent thatinhibits nuclear pore complex assembly, induces nuclear pore complexdisassembly, or inhibits nuclear pore complex function. In someinstances, the agent is a short hairpin RNA (shRNA). In some instances,the shRNA targets a nuclear pore complex component including, but notlimited to, Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153,Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, andELYS. In some instances, the shRNA reduces synthesis of a protein,thereby inhibiting nuclear pore complex assembly, inducing nuclear porecomplex disassembly, or inhibiting nuclear pore complex function.

MicroRNA (miRNA)

MicroRNA (miRNA) is a small non-coding RNA molecule (containing about 22nucleotides) found in plants, animals and some viruses that functions inRNA silencing and posttranscriptional regulation of gene expression.Encoded by eukaryotic nuclear DNA in plants and animals and by viral DNAin certain viruses whose genome is based on DNA, miRNAs function viabase-pairing with complementary sequences within mRNA molecules. As aresult, these mRNA molecules are silenced, by one or more of thefollowing processes: cleavage of the mRNA strand into two pieces,destabilization of the mRNA through shortening of its poly(A) tail, andless efficient translation of the mRNA into proteins by ribosomes. Insome embodiments, disclosed herein, the methods comprise an agent thatinhibits nuclear pore complex assembly, induces nuclear pore complexdisassembly, or inhibits nuclear pore complex function. In someinstances, the agent is a microRNA (miRNA). In some instances, the agentis a microRNA mimic. miRNA mimics contain non-natural or artificialdouble stranded miRNA-like RNA fragments. These RNA fragments areconstructed to contain a sequence motif on its 5′-end that is partiallycomplementary to the target sequence in the 3′UTR. Once these RNAfragments are introduced into cells the miRNA mimics binds specificallyto the targeted gene. The result is posttranscriptional repression ortranslational inhibition of the gene. In some instances, the miRNA ormiRNA mimic targets a nuclear pore complex component including, but notlimited to, Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153,Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, andELYS. In some instances, the miRNA or miRNA mimic reduces synthesis of aprotein, thereby inhibiting nuclear pore complex assembly, inducingnuclear pore complex disassembly, or inhibiting nuclear pore complexfunction.

Messenger RNA (mRNA)

Messenger RNA (mRNA) is a large family of RNA molecules that conveygenetic information from DNA to the ribosome, where they specify theamino acid sequence of the protein products of gene expression.Following transcription of primary transcript mRNA (known as pre-mRNA)by RNA polymerase, processed, mature mRNA is translated into a polymerof amino acids: a protein. As in DNA, mRNA genetic information is in thesequence of nucleotides, which are arranged into codons consisting ofthree base pairs each. Each codon encodes for a specific amino acid,except the stop codons, which terminate protein synthesis. In someembodiments, disclosed herein, the methods comprise an agent thatinhibits nuclear pore complex assembly, induces nuclear pore complexdisassembly, or inhibits nuclear pore complex function. In someinstances, the agent is a messenger RNA (mRNA). In some instances, themRNA has a mutated binding domain of a nuclear pore complex componentincluding, but not limited to, Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS, wherein the binding domain is non-functional but acts asa wild type, thereby outcompeting the wildtype mRNA and preventingstructural function. In some instances, the mRNA has a mutated bindingdomain of a nuclear pore complex component including, but not limitedto, Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62,Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155,Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS,wherein the binding domain is non-functional but acts as a wild type,thereby outcompeting the wildtype mRNA and preventing the transport ofthe component to the nuclear pore complex. In some instances, the mRNAhas a mutated regulatory domain of a nuclear pore complex componentincluding, but not limited to, Nup35, Nup37, Nup43, Nup45, Nup50, Nup54,Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107,Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2),Seh1, Sec13, NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR,GLE1, and ELYS, wherein the regulatory domain is non-functional but actsas a wild type, thereby outcompeting the wildtype mRNA and preventingstructural function. In some instances, the mRNA has a mutatedregulatory domain of a nuclear pore complex component including, but notlimited to, Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11),Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153,Nup155, Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13,NDC1, Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, andELYS, wherein the regulatory domain is non-functional but acts as a wildtype, thereby outcompeting the wildtype mRNA and preventing thetransport of the component to the nuclear pore complex. In someinstances, the mRNA outcompetes the wildtype mRNA of a protein of theprotein binding/interacting pairs listed in Table 1, thereby disruptingthe pair. In some instances, the mRNA outcompetes the wildtype mRNA of aprotein of the protein binding/interacting pairs listed in Table 2,thereby disrupting the pair. In some instances, the mRNA outcompetes thewildtype mRNA of a protein of the protein binding/interacting pairslisted in Table 3, thereby disrupting the pair. In some instances, themRNA reduces synthesis of a protein, thereby inhibiting nuclear porecomplex assembly, inducing nuclear pore complex disassembly, orinhibiting nuclear pore complex function.

GuideRNA (gRNA)

GuideRNA (gRNA) is a short synthetic RNA composed of a “scaffold”sequence necessary for Cas9-binding and a ˜20 nucleotide “spacer” or“targeting” sequence which defines the genomic target to be modified.CRISPR was originally employed to “knock-out” target genes in variouscell types and organisms, but modifications to the Cas9 enzyme haveextended the application of CRISPR to selectively activate or represstarget genes, purify specific regions of DNA, and even image DNA in livecells using fluorescence microscopy. In some embodiments, disclosedherein, the methods comprise an agent that inhibits nuclear pore complexassembly, induces nuclear pore complex disassembly, or inhibits nuclearpore complex function. In some instances, the agent is a guideRNA(gRNA). In some instances, the gRNA targets a nuclear pore complexcomponent including, but not limited to, Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS. In some instances, thegRNA reduces synthesis of a protein, thereby inhibiting nuclear porecomplex assembly, inducing nuclear pore complex disassembly, orinhibiting nuclear pore complex function.

Methods of Use

Disclosed herein, in some embodiments, is a method for treating aproliferative disease or disorder in an individual in need thereof,comprising: administering to the individual a therapeutically effectiveamount of an agent that inhibits nuclear pore complex assembly, inducesnuclear pore complex disassembly, or inhibits nuclear pore complexfunction. Further disclosed, in some embodiments, are pharmaceuticalcomposition, comprising (a) an agent that inhibits nuclear pore complexassembly, induces nuclear pore complex disassembly, or inhibits nuclearpore complex function; and (b) a pharmaceutically acceptable excipient.

In some instances, the agent inhibits expression of a component of thenuclear pore complex. In some instances, the agent promotes degradationof a component of the nuclear pore complex. In some instances, the agentinhibits function of a component of the nuclear pore complex. In someinstances, the agent modulates the expression of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsexpression of a regulator of the nuclear pore complex assembly. In someinstances, the agent modulates the function of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsfunction of a regulator of the nuclear pore complex assembly. In someinstances, the agent modulates the expression of a regulator of thenuclear pore complex disassembly. In some instances, the agent modulatesthe function of a regulator of the nuclear pore complex disassembly. Insome instances, the agent inhibits interaction between the components ofnuclear pore complex. In some instances, the agent inhibits therecruitment of nucleoporins to the nuclear pore complex. In someinstances, the agent inhibits the assembly of structure intermediates orpre-pores of nuclear pore complex. In some instances, the agent inhibitscell proliferation in malignant cells. In some instances, the agentinhibits cell proliferation in abnormally proliferative cells. In someinstances, the agent induces cell death in malignant cells. In someinstances, the agent induces cell death in abnormally proliferativecells.

In some instances, the agent inhibits Nup35, Nup37, Nup43, Nup45, Nup50,Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits Nup35, Nup37, Nup43,Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96,Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits Nup35 of the nuclear porecomplex. In some instances, the agent inhibits Nup37 of the nuclear porecomplex. In some instances, the agent inhibits Nup43 of the nuclear porecomplex. In some instances, the agent inhibits Nup45 of the nuclear porecomplex. In some instances, the agent inhibits Nup50 of the nuclear porecomplex. In some instances, the agent inhibits Nup54 of the nuclear porecomplex. In some instances, the agent inhibits Nup58(Nup11) of thenuclear pore complex. In some instances, the agent inhibits Nup62 of thenuclear pore complex. In some instances, the agent inhibits Nup75/85 ofthe nuclear pore complex. In some instances, the agent inhibits Nup93 ofthe nuclear pore complex. In some instances, the agent inhibits Nup96 ofthe nuclear pore complex. In some instances, the agent inhibits Nup107of the nuclear pore complex. In some instances, the agent inhibitsNup133 of the nuclear pore complex. In some instances, the agentinhibits Nup155 of the nuclear pore complex. In some instances, theagent inhibits Nup160 of the nuclear pore complex. In some instances,the agent inhibits Nup358(RanBP2) of the nuclear pore complex. In someinstances, the agent inhibits Seh1 of the nuclear pore complex. In someinstances, the agent inhibits Sec13 of the nuclear pore complex. In someinstances, the agent inhibits NDC1 of the nuclear pore complex. In someinstances, the agent inhibits Pom121 of the nuclear pore complex. Insome instances, the agent Nup210 of the nuclear pore complex. In someinstances, the agent inhibits RAE1 of the nuclear pore complex. In someinstances, the agent inhibits HCG1/CG1(Nup12) of the nuclear porecomplex. In some instances, the agent inhibits Aladin of the nuclearpore complex. In some instances, the agent inhibits TPR of the nuclearpore complex. In some instances, the agent inhibits GLE1 of the nuclearpore complex. In some instances, the agent inhibits ELYS of the nuclearpore complex. In some instances, the agent inhibits a component of thenuclear pore complex comprising a protein binding pair, therebydisrupting the pair. In some instances, the protein binding/interactingpair is selected from a pair listed in Table 1. In some instances, theprotein binding/interacting pair is selected from a pair listed in Table2. In some instances, the protein binding/interacting pair is selectedfrom a pair listed in Table 3.

In some instances, the agent is a small molecule. In some instances, theagent is a small interfering RNA (siRNA). In some instances, the agentis a short hairpin RNA (shRNA). In some instances, the agent is amicroRNA (miRNA). In some instances, the agent is a messenger RNA(mRNA). In some instances, the agent is a guideRNA (gRNA).

In some instances, the method further comprises administering anadditional therapeutic agent. In some instances, the agent and theadditional therapeutic agent are administered simultaneously. In someinstances, the agent and the additional therapeutic agent areadministered sequentially. In some instances, the agent is administeredbefore administering the additional therapeutic agent. In someinstances, the agent is administered after administering the additionaltherapeutic agent. In some instances, the additional therapeutic agentis an anti-proliferative agent. In some instances, the additionaltherapeutic agent is anti-cancer agent. In some instances, theadditional therapeutic agent is a chemotherapeutic agent. In someinstances, the additional therapeutic agent is a hormonal agent. In someinstances, the additional therapeutic agent is an anti-inflammatoryagent. In some instances, the additional therapeutic agent is a steroid.In some instances, the additional therapeutic agent is animmunotherapeutic agent. In some instances, the additional therapeuticagent is a targeted therapy agent.

In some instances, the agent and the additional therapeutic agent areadministered continuously for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 28,30 or more days. In some instances, the agent and the additionaltherapeutic agent are administered continuously for 1 or more days. Insome instances, the agent and the additional therapeutic agent areadministered continuously for 2 or more days. In some instances, theagent and the additional therapeutic agent are administered continuouslyfor 3 or more days. In some instances, the agent and the additionaltherapeutic agent are administered continuously for 4 or more days. Insome instances, the agent and the additional therapeutic agent areadministered continuously for 5 or more days. In some instances, theagent and the additional therapeutic agent are administered continuouslyfor 6 or more days. In some instances, the agent and the additionaltherapeutic agent are administered continuously for 7 or more days. Insome instances, the agent and the additional therapeutic agent areadministered continuously for 8 or more days. In some instances, theagent and the additional therapeutic agent are administered continuouslyfor 9 or more days. In some instances, the agent and the additionaltherapeutic agent are administered continuously for 10 or more days. Insome instances, the agent and the additional therapeutic agent areadministered continuously for 14 or more days. In some instances, theagent and the additional therapeutic agent are administered continuouslyfor 15 or more days. In some instances, the agent and the additionaltherapeutic agent are administered continuously for 28 or more days. Insome instances, the agent and the additional therapeutic agent areadministered continuously for 30 or more days.

In some cases, the agent and the additional therapeutic agent areadministered at predetermined time intervals for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 14, 15, 28, 30 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 1 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 2 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 3 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 4 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 5 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 6 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 7 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 8 or more days. In some instances the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 9 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 10 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 14 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 15 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 28 or more days. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 30 or more days.

In some embodiments, the agent and the additional therapeutic agent areadministered at predetermined time intervals for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 24, 36 or more months. In some instances, the agent andthe additional therapeutic agent are administered at predetermined timeintervals for 1 or more month. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 2 or more months. In some instances the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 3 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 4 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 5 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 6 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 7 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 8 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 9 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 10 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 11 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 12 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 24 or more months. In some instances, the agent and theadditional therapeutic agent are administered at predetermined timeintervals for 36 or more months.

In some cases, the agent and the additional therapeutic agent areadministered intermittently for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15,28, 30 or more days. In some instances, the agent and the additionaltherapeutic agent are administered intermittently for 1 or more days. Insome instances, the agent and the additional therapeutic agent areintermittently for 2 or more days. In some instances the agent and theadditional therapeutic agent are administered intermittently for 3 ormore days. In some instances, the agent and the additional therapeuticagent are administered intermittently for 4 or more days. In someinstances, the agent and the additional therapeutic agent areadministered intermittently for 5 or more days. In some instances, theagent and the additional therapeutic agent are administeredintermittently for 6 or more days. In some instances, the agent and theadditional therapeutic agent are administered intermittently for 7 ormore days. In some instances, the agent and the additional therapeuticagent are administered intermittently for 8 or more days. In someinstances, the agent and the additional therapeutic agent areadministered intermittently for 9 or more days. In some instances, theagent and the additional therapeutic agent are administeredintermittently for 10 or more days. In some instances, the agent and theadditional therapeutic agent are administered intermittently for 14 ormore days. In some instances, the agent and the additional therapeuticagent are administered intermittently for 15 or more days. In someinstances, the agent and the additional therapeutic agent areadministered intermittently for 28 or more days. In some instances, theagent and the additional therapeutic agent are administeredintermittently for 30 or more days.

In some instances, the agent is administered to an individual at atherapeutically effective amount. For example, the therapeuticallyeffective amount is optionally administered in 1 dose, 2 doses, 3 doses,4 doses, 5 doses, 6 doses or more. In some instances, thetherapeutically effective amount of an agent is administered to anindividual in 1 dose. In some instances, the therapeutically effectiveamount of an agent is administered to an individual in 2 or more doses.In some instances, the therapeutically effective amount of an agent isadministered an individual in 3 or more doses. In some instances, thetherapeutically effective amount of an agent is administered anindividual in 4 or more doses. In some instances, the therapeuticallyeffective amount of an agent is administered to an individual in 5 ormore doses. In some instances, the therapeutically effective amount ofan agent is administered an individual in 6 or more doses.

In some instances, the disease or disorder has abnormal nucleartransport. In some instances, the disease or disorder has normal nucleartransport. In some instances, the disease or disorder is a neoplasticdisease. In some instances, the disease or disorder is caused by amalignant cell. In some instances, the disease or disorder is aninflammatory disease. In some instances, the disease or disorder iscancer. In some instances, the cancer has a mutation in the Ras gene. Insome instances, the mutation is a G12D mutation. In some instances, theproliferative disease is atherosclerosis. In some instances, theproliferative disease is various forms of arthritis. In some instances,the proliferative disease is rheumatoid arthritis. In some instances,the proliferative disease is psoriasis. In some instances, theproliferative disease is various forms of fibrosis. In some instances,the proliferative disease is idiopathic pulmonary fibrosis. In someinstances, the proliferative disease is scleroderma. In some instances,the proliferative disease is cirrhosis of the liver. In some instances,the proliferative disease is benign prostatic hyperplasia. In someinstances, the proliferative disease is abnormal scar formation. In someinstances, the proliferative disease is inflammatory bowel disease.

Additional Therapeutic Agents

In some embodiments, one or more methods described herein furthercomprising administering an additional therapeutic agent. In someembodiments, the additional therapeutic agent comprises ananti-proliferative agent, chemotherapeutic agent, a hormonal agent, acytotoxin, a steroid, an immunotherapeutic agent, a targeted therapyagent, or an anti-inflammatory agent.

Chemotherapies as additional therapeutic agents include, but are notlimited to, hormone modulators, androgen receptor binding agents (e.g.,anti-androgens, bicalutamide, flutamide, nilutamide, MDV3100),gonadotropin-releasing hormone agonists and antagonists (e.g.,leuprolide, buserelin, histrelin, goserelin, deslorelin, nafarelin,abarelix, cetrorelix, ganirelix degarelix), androgen synthesisinhibitors (abiraterone, TOK-001), temozolomide, mitozolomide,dacarbazine, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, anthracyclines (e.g.,daunorubicin, doxorubicin, epirubicin, idarubicin), bleomycin,plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogenreceptor binding agents, cabazitaxel, paclitaxel, gemcitabine,navelbine, farnesyl-protein transferase inhibitors, transplatinum,5-fluorouracil, capecitabine, vincristin, vinblastin and methotrexate,topoisomerase inhibitors (e.g., irinotecan, topotecan, camptothecin,etoposide) or any derivative related agent of the foregoing. Many of theabove agents are also referred to as hormone therapy agents such as, forexample, androgen receptor binding agents, gonadotropin-releasinghormone agonists and antagonists, androgen synthesis inhibitors,estrogen receptor binding agents as well as aromatase inhibitors.

Immunotherapeutic agents generally rely on the use of immune effectorcells and molecules to target and destroy cancer cells. The immuneeffector may be, for example, a tumor antigen or an antibody specificfor some marker on the surface of a tumor cell. The tumor antigen orantibody alone may serve as an effector of therapy or it may recruitother cells to actually effect cell killing. An antibody also may beconjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin Achain, cholera toxin, pertussis toxin, etc.) and serve merely as atargeting agent. Alternatively, the effector may be a lymphocytecarrying a surface molecule that interacts, either directly orindirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells. Alternatively, a tumor antigen maystimulate a subject's immune system to target the specific tumor cellsusing cytotoxic T cells and NK cells. Immunotherapies include cancervaccines such as Sipuleucel-T, tumor-targeting antibodies such asbevacizumab and trastuzumab, T cell engagers, adoptive cell therapiesand the like.

Pharmaceutical Composition and Formulations

Disclosed herein, in some embodiments, are pharmaceutical composition,comprising (a) an agent that inhibits nuclear pore complex assembly,induces nuclear pore complex disassembly, or inhibits nuclear porecomplex function; and (b) a pharmaceutically acceptable excipient.

In certain embodiments, disclosed herein include pharmaceuticalcompositions and formulations comprising an agent described herein. Insome embodiments, the pharmaceutical compositions described herein areformulated for administering to a subject by systemic administration. Inother embodiments, the pharmaceutical compositions described herein areformulated for administering to a subject by local administration. Insome instances, the administration routes include, but are not limitedto, parenteral (e.g., intravenous, subcutaneous, intramuscular,intracerebral, intracerebroventricular, intra-articular,intraperitoneal, or intracranial), oral, sublingual, intranasal, buccal,rectal, or transdermal administration routes. In some instances, thepharmaceutical composition describe herein is formulated for parenteral(e.g., intravenous, subcutaneous, intramuscular, intracerebral,intracerebroventricular, intra-articular, intraperitoneal, orintracranial) administration. In other instances, the pharmaceuticalcomposition describe herein is formulated for oral administration. Inadditional instances, the pharmaceutical composition describe herein isformulated for sublingual administration. In additional instances, thepharmaceutical composition describe herein is formulated for intranasaladministration. In some cases, the pharmaceutical composition isadministered to a subject as an injection. In other instances, thepharmaceutical composition is administered to a subject as an infusion.

In some embodiments, the pharmaceutical formulations include, but arenot limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations (e.g., nanoparticleformulations), and mixed immediate and controlled release formulations.

In some embodiments, the pharmaceutical formulations include a carrieror carrier materials selected on the basis of compatibility with thecomposition disclosed herein, and the release profile properties of thedesired dosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. Pharmaceutically compatible carrier materials include, but are notlimited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins1999).

In some instances, the pharmaceutical formulations further include pHadjusting agents or buffering agents which include acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical formulation includes one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

In some instances, the pharmaceutical formulations further includediluent which are used to stabilize compounds because they can provide amore stable environment. Salts dissolved in buffered solutions (whichalso can provide pH control or maintenance) are utilized as diluents inthe art, including, but not limited to a phosphate buffered salinesolution. In certain instances, diluents increase bulk of thecomposition to facilitate compression or create sufficient bulk forhomogenous blend for capsule filling. Such compounds can include e.g.,lactose, starch, mannitol, sorbitol, dextrose, microcrystallinecellulose such as Avicel®; dibasic calcium phosphate, dicalciumphosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrouslactose, spray-dried lactose; pregelatinized starch, compressible sugar,such as Di-Pac® (Amstar); mannitol, hydroxypropylmethylcellulose,hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents,confectioner's sugar; monobasic calcium sulfate monohydrate, calciumsulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzedcereal solids, amylose; powdered cellulose, calcium carbonate; glycine,kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical formulations include disintegrationagents or disintegrants to facilitate the breakup or disintegration of asubstance. The term “disintegrate” include both the dissolution anddispersion of the dosage form when contacted with gastrointestinalfluid. Examples of disintegration agents include a starch, e.g., anatural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

In some instances, the pharmaceutical formulations include fillingagents such as lactose, calcium carbonate, calcium phosphate, dibasiccalcium phosphate, calcium sulfate, microcrystalline cellulose,cellulose powder, dextrose, dextrates, dextran, starches, pregelatinizedstarch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in thepharmaceutical formulations described herein for preventing, reducing orinhibiting adhesion or friction of materials. Exemplary lubricantsinclude, e.g., stearic acid, calcium hydroxide, talc, sodium stearylfumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetableoil such as hydrogenated soybean oil)(Sterotex®, higher fatty acids andtheir alkali-metal and alkaline earth metal salts, such as aluminum,calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol,talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate,sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™ sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, CabOSil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. Plasticizers canalso function as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyloleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

Stabilizers include compounds such as any antioxidation agents, buffers,acids, preservatives and the like.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetatecopolymer (S630), polyethylene glycol, e.g., the polyethylene glycol canhave a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitanmonolaurate, polyethoxylated sorbitan monolaurate, povidone and thelike.

Surfactants include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like.Additional surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. Sometimes, surfactants is included to enhance physicalstability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropylmethyl cellulose acetate stearate,hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol,alginates, acacia, chitosans and combinations thereof. Wetting agentsinclude compounds such as oleic acid, glyceryl monostearate, sorbitanmonooleate, sorbitan monolaurate, triethanolamine oleate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Therapeutic Regimens for a Pharmaceutical Composition

In some embodiments, a pharmaceutical compositions described herein areadministered for therapeutic applications. In some embodiments, thepharmaceutical composition is administered once per day, twice per day,three times per day or more. The pharmaceutical composition isadministered daily, every day, every alternate day, five days a week,once a week, every other week, two weeks per month, three weeks permonth, once a month, twice a month, three times per month, or more. Thepharmaceutical composition is administered for at least 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, ormore.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the composition is given continuously;alternatively, the dose of the composition being administered istemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). In some instances, the length of the drugholiday varies between 2 days and 1 year, including by way of exampleonly, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days,15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320days, 350 days, or 365 days. The dose reduction during a drug holiday isfrom 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained.

In some embodiments, the amount of a given agent that correspond to suchan amount varies depending upon factors such as the particular compound,the severity of the disease, the identity (e.g., weight) of the subjector host in need of treatment, but nevertheless is routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. In some instances, the desired dose is conveniently presentedin a single dose or as divided doses administered simultaneously (orover a short period of time) or at appropriate intervals, for example astwo, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesis altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

In some embodiments, toxicity and therapeutic efficacy of suchtherapeutic regimens are determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, including, but notlimited to, the determination of the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between the toxic and therapeuticeffects is the therapeutic index and it is expressed as the ratiobetween LD50 and ED50. Compounds exhibiting high therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for use in human. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with minimal toxicity. The dosagevaries within this range depending upon the dosage form employed and theroute of administration utilized.

Screening for Agents

Disclosed herein, in some embodiments, are methods of screening for atherapeutic agent for treating a proliferative disease or disorder in anindividual in need thereof, the method comprising: (a) contacting a cellwith a test agent; (b) detecting (i) inhibition of nuclear pore complexassembly as compared to a control; or (ii) induction of nuclear porecomplex disassembly as compared to a control; and (c) identifying thetest agent as a therapeutic agent if the test agent reduces the numberof nuclear pore complexes in the cell as compared to the control.

In some instances, the cell is a tumor cell in vitro. In some instances,the cell is a tumor cell in vivo. In some instances, step (b) comprisesperforming: (i) cell proliferation or survival assay in vitro; and (ii)tumor formation, growth or metastasis assay in vivo.

In some instances, the in vivo assay is a Protein-fragmentcomplementation assay, or PCA. In some instances, the in vivo assay is aBimolecular Fluorescence Complementation (BiFC) assay. In someinstances, the in vivo assay is a Fluorescence resonance energy transfer(FRET) assay.

In some instances, the detection is by direct visualization. In someinstances, the detection is by microscopy imaging, Western blot,immunohistochemistry, ELISA, SPARCL, fluorescent signal detector,chromatography, radioactive binding assay, a fluorescence binding assay,a kinetic exclusion assay, a crystallography assay, or live imaging.

In some instances, the agent inhibits expression of a component of thenuclear pore complex. In some instances, the agent promotes degradationof a component of the nuclear pore complex. In some instances, the agentinhibits function of a component of the nuclear pore complex. In someinstances, the agent modulates the expression of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsexpression of a regulator of the nuclear pore complex assembly. In someinstances, the agent modulates the function of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsfunction of a regulator of the nuclear pore complex assembly. In someinstances, the agent modulates the expression of a regulator of thenuclear pore complex disassembly. In some instances, the agent modulatesthe function of a regulator of the nuclear pore complex disassembly. Insome instances, the agent inhibits interaction between the components ofnuclear pore complex. In some instances, the agent disrupts a proteinbinding/interacting pair comprising pairs listed in Table 1. In someinstances, the agent disrupts a protein binding/interacting paircomprising pairs listed in Table 2. In some instances, the agentdisrupts a protein binding/interacting pair comprising pairs listed inTable 3. In some instances, the agent inhibits synthesis of a componentof the nuclear pore complex, thereby inhibiting nuclear pore complexassembly, inducing nuclear pore complex disassembly, or inhibitingnuclear pore complex function. In some instances, the agent disrupts thetransport of the nucleoporins. In some instances, the agent inhibits therecruitment of nucleoporins to the nuclear pore complex. In someinstances, the agent inhibits the assembly of structure intermediates orpre-pores of nuclear pore complex. In some instances, the agent inhibitscell proliferation in malignant cells. In some instances, the agentinhibits cell proliferation in abnormally proliferative cells. In someinstances, the agent induces cell death in malignant cells. In someinstances, the agent induces cell death in abnormally proliferativecells.

In some instances, the agent inhibits expression or function of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88,Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188,Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits Nup35, Nup37, Nup43,Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96,Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits Nup35 of the nuclear porecomplex. In some instances, the agent inhibits Nup37 of the nuclear porecomplex. In some instances, the agent inhibits Nup43 of the nuclear porecomplex. In some instances, the agent inhibits Nup45 of the nuclear porecomplex. In some instances, the agent inhibits Nup50 of the nuclear porecomplex. In some instances, the agent inhibits Nup54 of the nuclear porecomplex. In some instances, the agent inhibits Nup58(Nup11) of thenuclear pore complex. In some instances, the agent inhibits Nup62 of thenuclear pore complex. In some instances, the agent inhibits Nup75/85 ofthe nuclear pore complex. In some instances, the agent inhibits Nup93 ofthe nuclear pore complex. In some instances, the agent inhibits Nup96 ofthe nuclear pore complex. In some instances, the agent inhibits Nup107of the nuclear pore complex. In some instances, the agent inhibitsNup133 of the nuclear pore complex. In some instances, the agentinhibits Nup155 of the nuclear pore complex. In some instances, theagent inhibits Nup160 of the nuclear pore complex. In some instances,the agent inhibits Nup358(RanBP2) of the nuclear pore complex. In someinstances, the agent inhibits Seh1 of the nuclear pore complex. In someinstances, the agent inhibits Sec13 of the nuclear pore complex. In someinstances, the agent inhibits NDC1 of the nuclear pore complex. In someinstances, the agent inhibits Pom121 of the nuclear pore complex. Insome instances, the agent Nup210 of the nuclear pore complex. In someinstances, the agent inhibits RAE1 of the nuclear pore complex. In someinstances, the agent inhibits HCG1/CG1(Nup12) of the nuclear porecomplex. In some instances, the agent inhibits Aladin of the nuclearpore complex. In some instances, the agent inhibits TPR of the nuclearpore complex. In some instances, the agent inhibits GLE1 of the nuclearpore complex. In some instances, the agent inhibits ELYS of the nuclearpore complex. In some instances, the agent inhibits binding of acomponent of the nuclear pore complex to other components of the nuclearpore complex. In some instances, the agent inhibits a component of thenuclear pore complex comprising a protein binding pair, therebydisrupting the pair. In some instances, the protein binding pair isselected from a pair listed in Table 1. In some instances, the proteinbinding pair is selected from a pair listed in Table 2. In someinstances, the protein binding pair is selected from a pair listed inTable 3. In some instances, the therapeutic agent targets the RNA or DNAof Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62,Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155,Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS ofthe nuclear pore complex. In some instances, the therapeutic agent bindsto Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62,Nup75/85, Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155,Nup160, Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1,Pom121, Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS ofthe nuclear pore complex.

In some instances, the therapeutic agent is a small molecule. In someinstances, the therapeutic agent is a small interfering RNA (siRNA). Insome instances, the therapeutic agent is a short hairpin RNA (shRNA). Insome instances, the therapeutic agent is a microRNA (miRNA). In someinstances, the therapeutic agent is a messenger RNA (mRNA). In someinstances, the therapeutic agent is a guideRNA (gRNA). In someinstances, the therapeutic agent is an antisense oligonucleotide. Insome instances, the therapeutic agent is a peptide. In some instances,the therapeutic agent is a peptidomimetic. In some instances, thetherapeutic agent is an aptamer.

In some instances, the disease or disorder has abnormal nucleartransport. In some instances, the disease or disorder has normal nucleartransport. In some instances, the disease or disorder is a neoplasticdisease. In some instances, the disease or disorder is caused by amalignant cell. In some instances, the disease or disorder is aninflammatory disease. In some instances, the disease or disorder iscancer. In some instances, the cancer has a mutation in the Ras gene. Insome instances, the mutation is a G12D mutation. In some instances,cancer with G12D mutation is more sensitive to the agent. In someinstances, the proliferative disease is atherosclerosis. In someinstances, the proliferative disease is various forms of arthritis. Insome instances, the proliferative disease is rheumatoid arthritis. Insome instances, the proliferative disease is psoriasis. In someinstances, the proliferative disease is various forms of fibrosis. Insome instances, the proliferative disease is idiopathic pulmonaryfibrosis. In some instances, the proliferative disease is scleroderma.In some instances, the proliferative disease is cirrhosis of the liver.In some instances, the proliferative disease is benign prostatichyperplasia. In some instances, the proliferative disease is abnormalscar formation. In some instances, the proliferative disease isinflammatory bowel disease.

Disclosed herein, in some embodiments, are methods of screening for atherapeutic agent for treating a proliferative disease or disorder in anindividual in need thereof, the method comprising: (a) contacting a cellwith a test agent; (b) detecting inhibition of nuclear transport ascompared to a control; and (c) identifying the test agent as atherapeutic agent if the test agent reduces the expression or activityof a component of nuclear pore complex in the cell as compared to thecontrol.

In some instances, the cell is a tumor cell in vitro. In some instances,the cell is a tumor cell in vivo. In some instances, step (b) comprisesperforming: (i) cell proliferation or survival assay in vitro; and (ii)tumor formation, growth or metastasis assay in vivo.

In some instances, the expression or activity of the component ismeasured by microscopy imaging, Western blot, immunohistochemistry,ELISA, SPARCL, fluorescent signal detector, chromatography, radioactivebinding assay, a fluorescence binding assay, a kinetic exclusion assay,a crystallography assay, PCR, or gel electrophoresis.

In some instances, the agent inhibits expression of a component of thenuclear pore complex. In some instances, the agent promotes degradationof a component of the nuclear pore complex. In some instances, the agentinhibits function of a component of the nuclear pore complex. In someinstances, the agent modulates the expression of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsexpression of a regulator of the nuclear pore complex assembly. In someinstances, the agent modulates the function of a regulator of thenuclear pore complex assembly. In some instances, the agent inhibitsfunction of a regulator of the nuclear pore complex assembly. In someinstances, the agent modulates the expression of a regulator of thenuclear pore complex disassembly. In some instances, the agent modulatesthe function of a regulator of the nuclear pore complex disassembly. Insome instances, the agent inhibits interaction between the components ofnuclear pore complex. In some instances, the agent inhibits therecruitment of nucleoporins to the nuclear pore complex. In someinstances, the agent inhibits the assembly of structure intermediates orpre-pores of nuclear pore complex. In some instances, the agent inhibitscell proliferation in malignant cells. In some instances, the agentinhibits cell proliferation in abnormally proliferative cells. In someinstances, the agent induces cell death in malignant cells. In someinstances, the agent induces cell death in abnormally proliferativecells.

In some instances, the agent inhibits Nup35, Nup37, Nup43, Nup45, Nup50,Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the agent inhibits Nup35, Nup37, Nup43,Nup45, Nup50, Nup54, Nup58, Nup62, Nup75/85, Nup93, Nup96, Nup107,Nup133, Nup155, Nup160, Nup358, Seh1, Sec13, NDC1, Pom121, Nup210, Rae1,HCG1/CG1, Aladin, TPR, or Elys of the nuclear pore complex. In someinstances, the agent inhibits Nup35 of the nuclear pore complex. In someinstances, the agent inhibits Nup37 of the nuclear pore complex. In someinstances, the agent inhibits Nup43 of the nuclear pore complex. In someinstances, the agent inhibits Nup45 of the nuclear pore complex. In someinstances, the agent inhibits Nup50 of the nuclear pore complex. In someinstances, the agent inhibits Nup54 of the nuclear pore complex. In someinstances, the agent inhibits Nup58 of the nuclear pore complex. In someinstances, the agent inhibits Nup62 of the nuclear pore complex. In someinstances, the agent inhibits Nup75/85 of the nuclear pore complex. Insome instances, the agent inhibits Nup93 of the nuclear pore complex. Insome instances, the agent inhibits Nup96 of the nuclear pore complex. Insome instances, the agent inhibits Nup107 of the nuclear pore complex.In some instances, the agent inhibits Nup133 of the nuclear porecomplex. In some instances, the agent inhibits Nup155 of the nuclearpore complex. In some instances, the agent inhibits Nup160 of thenuclear pore complex. In some instances, the agent inhibits Nup358 ofthe nuclear pore complex. In some instances, the agent inhibits Seh1 ofthe nuclear pore complex. In some instances, the agent inhibits Sec13 ofthe nuclear pore complex. In some instances, the agent inhibits NDC1 ofthe nuclear pore complex. In some instances, the agent inhibits Pom121of the nuclear pore complex. In some instances, the agent Nup210 of thenuclear pore complex. In some instances, the agent inhibits Rae1 of thenuclear pore complex. In some instances, the agent inhibits HCG1/CG1 ofthe nuclear pore complex. In some instances, the agent inhibits Aladinof the nuclear pore complex. In some instances, the agent inhibits TPRof the nuclear pore complex. In some instances, the agent inhibits Elysof the nuclear pore complex. In some instances, the agent inhibits acomponent of the nuclear pore complex comprising a protein binding pair,thereby disrupting the pair. In some instances, the proteinbinding/interacting pair is selected from a pair listed in Table 1. Insome instances, the protein binding/interacting pair is selected from apair listed in Table 2. In some instances, the proteinbinding/interacting pair is selected from a pair listed in Table 3. Insome instances, the therapeutic agent targets the RNA or DNA of Nup35,Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88,Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188,Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex. In some instances, the therapeutic agent binds to Nup35, Nup37,Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93,Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205,Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS of the nuclear porecomplex.

In some instances, the therapeutic agent is a small molecule. In someinstances, the therapeutic agent is a small interfering RNA (siRNA). Insome instances, the therapeutic agent is a short hairpin RNA (shRNA). Insome instances, the therapeutic agent is a microRNA (miRNA). In someinstances, the therapeutic agent is a messenger RNA (mRNA). In someinstances, the therapeutic agent is a guideRNA (gRNA). In someinstances, the therapeutic agent is an antisense oligonucleotide. Insome instances, the therapeutic agent is a peptide. In some instances,the therapeutic agent is a peptidomimetic. In some instances, thetherapeutic agent is an aptamer.

In some instances, the disease or disorder has abnormal nucleartransport. In some instances, the disease or disorder has normal nucleartransport. In some instances, the disease or disorder is a neoplasticdisease. In some instances, the disease or disorder is caused by amalignant cell. In some instances, the disease or disorder is aninflammatory disease. In some instances, the disease or disorder iscancer. In some instances, the cancer has a mutation in the Ras gene. Insome instances, the mutation is a G12D mutation. In some instances,cancer with G12D mutation is more sensitive to the agent. In someinstances, the proliferative disease is atherosclerosis. In someinstances, the proliferative disease is various forms of arthritis. Insome instances, the proliferative disease is rheumatoid arthritis. Insome instances, the proliferative disease is psoriasis. In someinstances, the proliferative disease is various forms of fibrosis. Insome instances, the proliferative disease is idiopathic pulmonaryfibrosis. In some instances, the proliferative disease is scleroderma.In some instances, the proliferative disease is cirrhosis of the liver.In some instances, the proliferative disease is benign prostatichyperplasia. In some instances, the proliferative disease is abnormalscar formation. In some instances, the proliferative disease isinflammatory bowel disease.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods described herein. Such kitsinclude a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. In one embodiment, thecontainers are formed from a variety of materials such as glass orplastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include a therapeutic agent disclosedherein, optionally with one or more additional therapeutic agentsdisclosed herein. Such kits optionally include an identifyingdescription or label or instructions relating to its use in the methodsdescribed herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1. Nuclear Pore Complex Assembly or Disassembly Assay in Animals

Animals, including but not limited to, mice, rats, fish are treated withnuclear pore complex (NPC) assembly and disassembly regulators includingbut not limited to small molecules, siRNAs, shRNAs, microRNAs, mRNAs,gRNA/CRISPR. The number of NPCs is analyzed in isolated tissues, cellsand nuclei by directly measuring the amount of NPCs on nuclear membranesor the levels of NPC proteins or RNA in tissue or cell extracts.

The measurement of NPCs on nuclear membranes is performed in fixed orunfixed isolated cells, tissues, or nuclei. Fixed refers to treatment ofcells, tissues or isolated nuclei with a fixative agent (such asformaldehyde, paraformaldehyde, glutaraldehyde, methanol, acetone, orother) before analysis. Visualization and quantification of NPCnumber/levels in the nuclear membranes of cells, tissues or nuclei isperformed by direct or indirect antibody detection-coupled to a methodto measure antibody levels such as microscopy imaging, ELISA, SPARCL,fluorescent signal detector. In the assay, primary antibodies thatrecognize one or more NPC components (including, but not limited to,Nup35, Nup37, Nup43, Nup45, Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85,Nup88, Nup93, Nup96, Nup98, Nup107, Nup133, Nup153, Nup155, Nup160,Nup188, Nup205, Nup214, Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121,Nup210, RAE1, HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS) areemployed. Primary antibodies are directly labeled (direct assay) or usedin combination with secondary detection systems such as secondaryantibodies, biotin/streptavidin system, lumio.

For the analysis of nuclear pore assembly in cell, tissue or nucleiextracts, whole protein extracts or nuclear membrane protein extractsare prepared from tissues or cells of control and candidate-treatedanimals and the assembly of NPCs is determined by measuring proteinlevels for specific nuclear pore components. Protein levels aredetermined by western blot assays, ELISA, SPARCL, chromatography, massspectrometry, or other means of quantifying specific protein levels.

To indirectly assay NPC assembly by measuring RNA levels, total RNA ormRNA is prepared from tissues or cells of treated animals and theexpression levels of NPC components is determined by PCR, sequencing,hybridization methods, or other assays that allow to quantify specificRNA molecules.

Example 2. Nuclear Pore Complex Assembly or Disassembly Assay inCultured Cells

The assembly or disassembly of nuclear pore complexes (NPCs) is followedby visualization of NPCs in the nuclear membranes of live or fixed cellsor by measuring protein or RNA levels in cell extracts.

Live Cell Assays:

For live cells, the fluorescent signal of an endogenous NPC componenttagged with a fluorescent marker (e.g GFP, RFP, Tomato or any otherfluorescent tag), or of an ectopically expressed fluorescently-taggedNPC component, is used to visualize and quantify the number of NPCs atthe nuclear membrane by the use of microscopy imaging. The increase ordecrease in the signal of NPCs in cells is employed to discovermolecules that activate or inhibit NPC assembly.

Dividing cells expressing an endogenously tagged NPC component(generated by inserting a fluorescent tag in frame with the endogenousgene of NPCs member e.g. by using CRISPR technology), or dividing cellsectopically expressing tagged NPC component (such as a cell linegenerated by insertion of a plasmid carrying the nuclear pore componentfused in frame with a fluorescent tag) are grown in multi-well cultureplates at low density. Cells are incubated with potential regulators ofNPC assembly and disassembly (small chemical entities, siRNA, shRNAs,microRNAs, mRNA, gRNA/CRISPR, or others) for 2-5 days and quantifiedusing microscopy imaging (epifluorescent microscope, confocalmicroscope, spinning disk microscope, light sheet microscope, orsimilar) or other methods of measuring fluorescent signals. The signalfor NPCs in cells is quantified using computer software.

Fixed Cell Assays:

For fixed cells, the amount of NPCs is determined by direct or indirectimmunofluorescence using antibodies that recognize one or more NPCcomponents (including, but not limited to, Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS) or antibodies thatrecognize the tag fused to the NPC component (fluorescent tag such asGFP, RFP, CFP, TOMATO, antibody tag e.g HA, MYC, FLAG, or others e.g.biotin, lumino, etc). Primary antibodies are labeled with a fluorescentmolecule (e.g. FITC, CY5, Alexa fluor, quantum dots) or used incombination with labeled secondary antibodies or other chemical entitiesused for detection (such as streptavidin). Quantification of NPC numberin cells is performed by microscopy imaging, ELISA, SPARCL, fluorescentsignal detector, or other means of detecting antibodies, light orchemical signals.

Dividing cells expressing an endogenously tagged NPC component(generated by inserting a fluorescent tag in frame with the endogenousgene of NPCs member e.g by CRISPR technology), or dividing cellsectopically expressing a tagged NPC component (such as a cell linegenerated by insertion of a plasmid carrying the nuclear pore componentfused in frame with a fluorescent tag) are grown in multi-well cultureplates at low density. Cells are incubated with potential regulators ofNPC assembly and disassembly (small chemical entities, siRNA, shRNAs,microRNAs, mRNA, gRNA/CRISPR, or others) for 2-5 days and fixed. Thenumber of NPCs is quantified using microscopy imaging (epifluorescentmicroscope, confocal microscope, spinning disk microscope, light sheetmicroscope, or similar) or other methods of measuring fluorescentsignals. The signal for NPCs in cells is quantified using computersoftware. Alternatively, antibodies that recognize the tag fused to theNPC component (fluorescent tag such as GFP, RFP, CFP, TOMATO, antibodytag e.g HA, MYC, FLAG, or others e.g. biotin, lumino, etc). are usedafter fixation to enhance the NPC fluorescent signal. Primary antibodiesare labeled with a fluorescent molecule (e.g. FITC, CY5, Alexa fluor,quantum dots) or used in combination with labeled secondary antibodiesor other chemical entities used for detection (such as streptavidin).Quantification of NPC number in cells is performed by microscopyimaging, ELISA, SPARCL, fluorescent signal detector, or other means ofdetecting antibodies, light or chemical signals.

Alternatively, unmodified dividing cells are grown for 2-5 days in thepresence of potential regulators of NPC assembly (small chemicalentities, siRNA, shRNAs, microRNAs, mRNAs, gRNA/CRISPR, or others).Cells are fixed and nuclear complexes are analyzed either by directimmunofluorescence using primary antibodies that recognize a NPCcomponent (including, but not limited to, Nup35, Nup37, Nup43, Nup45,Nup50, Nup54, Nup58(Nup11), Nup62, Nup75/85, Nup88, Nup93, Nup96, Nup98,Nup107, Nup133, Nup153, Nup155, Nup160, Nup188, Nup205, Nup214,Nup358(RanBP2), Seh1, Sec13, NDC1, Pom121, Nup210, RAE1,HCG1/CG1(Nup12), Aladin, TPR, GLE1, and ELYS) labeled with a fluorescentdye; or indirect immunofluorescence using a secondary antibody or otherchemical entities used for detection (such as streptavidin).Alternatively, cell proliferation and cell death is also measured as aresult of inhibition of nuclear pore assembly.

Cell Extracts:

For the analysis of nuclear pore assembly in cell extracts, whole cellor nuclear membrane protein extracts are prepared from cells treatedwith control and candidate molecules and the assembly of NPCs isfollowed by western blot assays, ELISA, SPARCL, chromatography, massspectrometry, or any other mean of quantifying specific protein levels.

Dividing cells are grown for 2-5 days in the presence of potentialregulators of NPC assembly (small chemical entities, siRNA, shRNAs,microRNAs, mRNAs, gRNA/CRISPR, or others). Cells are collected and wholecell or nuclear membrane protein extracts are prepared usingconventional protein extraction methods. The assembly of NPCs isfollowed by western blot assays, ELISA, SPARCL, chromatography, massspectrometry, or any other mean of quantifying specific protein levels.

To assay NPC assembly by measuring RNA levels, total RNA or mRNA isprepared from treated cells and the expression levels of NPC componentsis determined by PCR, RNA sequencing, hybridization methods, or otherassays that allow to quantify specific RNA molecules.

Example 3. Indirect Nuclear Pore Complex Assembly Assay In Vitro

Nuclear pore complex (NPC) assembly relies on the interaction ofdifferent components of the structure and their recruitment to thenuclear membranes. Preventing the interaction between nucleoporins alsoinhibits NPC assembly. To identify molecules that prevent theinteraction or association of NPC components, native or taggednucleoporin proteins are produced in bacteria, yeast, mammalian cells,cell-free transcription/translation systems, or other systems that allowrecombinant protein production and purified using conventionalpurification methods (such as liquid chromatography, affinitypurification columns, etc). Purified nucleoporins are combined withpotential regulators of protein-protein interaction (e.g. smallmolecules) and the association dissociation of protein complexes isdetermined using standard protein-protein interaction detection methods,such as methods based on crystal resonance or quartz crystalmicrobalance (QCM), surface plasmon resonance (SPR, e.g. Biacore),scanning tunneling microscopy (STM), total internal reflectionfluorescence (TIRF), isothermal titration calorimetry (ITC),differential scanning calorimetry (DSC), fluorescence resonance energytransfer, atomic force microscopy (AFM), or any other method that allowsmeasuring protein-protein interactions. Alternatively, interactionbetween nucleoporins is measured in cell extracts instead of isolatedproteins.

Example 4. Indirect Nuclear Pore Complex Assembly Assay In Vivo

Nuclear pore complex (NPC) assembly relies on the interaction ofdifferent components of the structure and their recruitment to thenuclear membranes. Preventing the interaction between nucleoporins alsoinhibits NPC assembly. To identify molecules that prevent theinteraction or association of NPC components in vivo, interactingnucleoporin proteins or nucleoporin protein fragments fused to tags thatallow to detect their association are produced in bacteria, yeast,mammalian cells, multicellular organisms (such as C. elegans, Zebrafish,Drosophila) or other systems that allow ectopic protein expression.Cells are combined with potential regulators of protein-proteininteraction (e.g. small molecules) and the association/dissociation ofprotein complexes is determined using standard protein-proteininteraction detection methods, such as methods based on Fluorescenceresonance energy transfer (FRET), Bioluminescence Resonance EnergyTransfer (BRET), Bimolecular Fluorescence Complementation (BiFC),Protein-fragment complementation assay (PCA) or any other method thatallows measuring protein-protein interactions in cells. Protein tagsthat are combined with nucleoporin proteins or nucleoporin proteinfragments include, but are not limited to, fluorescent tags that allowenergy transfer for FRET or BRET (EBFP2, EGFP, ECFP, EYFP, Cerulean,Venus, MiCy, mKO, TFP1, mVenus, CyPet, YPet, mCherry, Venus, tdTomato,mPlum, TagBFP, TagGFP2, TagGFP2, TagRFP, TurboFP, mOrange Luciferase,NanoLuc), or split proteins for BIFC or PCA (split-Beta-lactamase,split-Dihydrofolate reductase (DHFR), split-Focal adhesion kinase (FAK),split-Gal4 transcription factor, split-GFP, split-YFP, split-CFP,split-Horseradish peroxidase (HRP), split-Infrared fluorescent proteinIFP1.4, split-LacZ beta-galactosidase, split-Luciferase, recombinaseenhanced bimolecular luciferase (ReBiL), NanoLuc, and NanoBIT, split-TEV(Tobacco etch virus protease) and split-Ubiquitin). Alternatively,interaction between nucleoporins is measured in cell extracts instead oflive cells proteins.

Example 5. Nuclear Pore Complex Component Depletion in Cultured Cells

A375 cells were infected with lentivirus carrying Control, Nup160 orNDC1 shRNAs and selected to obtain stable cell lines (FIG. 2A), ortransfected with Control or Nup160 specific siRNAs (FIG. 2B). Cells wereallowed to proliferate for 3 days, fixed and stained with antibodiesagainst different nuclear pore components. mAb414 recognizes Nup358,Nup214, Nup153 and Nup62. Anti-Nup358 recognizes Nup358.

Example 6. Inhibition of Nuclear Pore Complex Assembly with shRNAs

A375 cells were infected with lentivirus carrying inducible Control,Nup160 or NDC1 shRNAs and selected to obtain stable cell lines. Cellswere allowed to grow to confluency and shRNA was induced withdoxycycline for 48 hours. Cells were either replated at 1:10 dilution toallow proliferation (FIG. 3A), or kept at confluency (FIG. 3B), and cellnumber was measured for 8 days.

Example 7. Inhibition of Nuclear Pore Complex Assembly with shRNAs

Proliferating or post-mitotic cells were treated with control or Nup160shRNAs to inhibit nuclear pore assembly and stained with antibodiesagainst myotubes (MHC) and nuclear pore complexes (Nup358). Hoechst wasused as nuclear marker. Cell proliferation was slowed down by growingcells in low-serum media and cell number was measured over time. Controlor Nup160 shRNAs were induced at time 0.

Example 8. Cell Cycle Analysis of Cells with Inhibited Nuclear PoreComplex Assembly

A375 cells expressing inducible Control or Nup160 shRNAs weresynchronized for 16 hours in the presence of doxycycline to induce shRNAexpression. Cells were released, allowed to grow for 72 hours andstained with Hoechst. Cell cycle progression was analyzed by flowcytometry.

Example 9. Cell Cycle Analysis of Cells with Inhibited Nuclear PoreComplex Assembly

A375 cells expressing tetracycline-inducible Control, Nup160 or NDC1shRNAs were synchronized for 16 hours in the presence of doxycycline toinduce shRNA expression. Cells were released, allowed to grow for 24, 72or 96 hours and stained with Hoechst. Cell cycle progression wasanalyzed by flow cytometry.

Example 10. Apoptosis in Cells with Inhibited Nuclear Pore ComplexAssembly

A375 cells expressing tetracycline-inducible Control, Nup160 or NDC1shRNAs were synchronized for 16 hours in the presence of doxycycline toinduce shRNA expression. Cells were released, allowed to grow. Anexin V(FIG. 7A-7B) and cell death marker (FIG. 7C) staining coupled to flowcytometry was used to determine the percentage of apoptotic and viablecells at different times after induction of knockdown.

Example 11. Cell Proliferation and Cell Death in Cancer Cells withInhibited Nuclear Pore assembly

Different cancer cell lines were infected with control or Nup160 shRNAsto block nuclear pore assembly and cell number was measured over time.All cell lines are human with the exception of B16/F10 which is mouse.B16/F10 cells were transfected with control or Nup160 siRNAs and cellproliferation was determined by measuring cell number over time.

Example 12. Tumor Growth in Cancer Cells with Inhibited Nuclear PoreAssembly

HT-29 colorectal cancer cells carrying tetracycline inducible control orNup160 shRNAs were injected subcutaneously in NOD-SCID immunodeficientmice. Tumors were allowed to grow to 50-100 mm³ and shRNA was induced byfeeding the animals with doxycycline on Day 14. Tumor volume wasmeasured over time and/or at the end of treatment.

After treatment, tumors were isolated, sectioned and stained with anantibody against the nuclear pore complex component Nup98 and withHoechst to stain the nucleus.

Example 13. Tumor Growth in Cancer Cells with Inhibited Nuclear PoreAssembly

A375 skin melanoma cancer cells carrying tetracycline inducible controlor Nup160 shRNAs were injected subcutaneously in NOD-SCIDimmunodeficient mice. Tumors were allowed to grow to 50-100 mm³ andshRNA was induced by feeding the animals with doxycycline on Day 9.Tumor volume or weight was measured over time.

Example 14. Effect of Inhibition of Nuclear Pore Complex Assembly onCancer and Normal Cells

A375 melanoma cancer cells and IMR90 normal fibroblasts were infectedwith lentivirus carrying inducible Control or Nup160 shRNAs (N1) andselected to obtain stable cell lines. shRNA expression was induced withdoxycycline and cell number was determine over the course of 7 days.

Example 15. Cancer Cells Carrying Ras Mutation are More Sensitive toInhibition of Nuclear Pore Assembly

DLD-1 colorectal cancer cells carrying wild type or mutated Ras (G12D)were infected with lentivirus carrying inducible Control or Nup160shRNAs and selected to obtain stable cell lines. shRNA expression wasinduced with doxycycline and cell number was determine over the courseof 7 days.

Example 16. Inhibition of Nuclear Pore Complex Assembly

Dividing cells expressing Control or Nup160 inducible shRNAs were seededat low density and shRNA expression was induced by doxycycline. Cellswere allowed to grow and divide and nuclear pore complex were stainedwith two different antibodies. FIG. 23 illustrates a detectablereduction in nuclear pore complex signal when nuclear pore complexassembly is inhibited. Nuclear pore complex inhibition is followed byquantifying the decrease in the signal at the nuclear periphery (FIG. 10) and/or by increase in the cytoplasmic aggregates that result fromnuclear pore complex assembly inhibition.

Example 17. Tagging Endogenous Nucleoporins

Cells expressing an endogenous nuclear pore complex component taggedwith GFP using CRISPR technology were imaged by confocal microscopy.Tagging of endogenous nucleoporins is used to detect nuclear porecomplexes in the screen for nuclear pore complex assembly regulatorsinstead of using antibodies. Using cell lines with endogenously taggednuclear pore complex components also allow for screens to be performedin live cells.

Example 18. Analysis of Tumor Formation, Growth, Metastasis andRemission in Mice

In these assays cancer formation is induced in mice and cancerdevelopment (tumor growth and metastasis) in control or treated mice isfollowed over time. For cancer induction, mice are injected with cancercells subcutaneously or in specific tissues, such as liver or blood.Alternatively, tumors are induced chemically (e.g. as liver tumors withDEN, Aflatoxin or CCL4), or genetic models of cancer (e.g. p53 knockout,Ink4a/Arf knockout, PTEN knockout, activating kRas (G12D) mutantexpressing mice or Myc oncogene expressing mice) are used to inducetumors in mice. Animals that develop cancer are treated with nuclearpore complex (NPC) assembly and disassembly regulators including but notlimited to small molecules, siRNAs, shRNAs, microRNAs, mRNAs,gRNA/CRISPR and tumor growth is followed over time by measuring tumorsize (or by blood analyses for blood malignancies such as leukemias andlymphomas). Alternatively, cancer cells expressing a luciferase oranother bioluminescent reporter or fluorescent reporter are used togenerate tumors and tumor growth and metastasis are followed by in vivooptical imaging such as Xenogen, IVIS® Spectrum, NightOWL LB 983, CRiMaestro 2, or other animal imaging systems. After treatment, tumors arecollected, weighted, and the number of NPCs is analyzed in isolatedtissues, cells and nuclei from the tumors by microscopy analysis usingantibodies against nuclear pore complex component or by measuring theprotein or RNA levels of nuclear pore complex proteins in tumors or cellextracts.

Example 19. In Vivo Analysis of Tumor Development

In these assays mice are injected with cancer cells subcutaneously or inspecific tissues, such as liver or blood. Alternatively, tumors areinduced chemically (e.g. as liver tumors with DEN, Aflatoxin or CCL4),or genetic models of cancer (e.g. p53 knockout, Ink4a/Arf knockout, PTENknockout, activating kRAS (G12D) mutant expressing mice or Myc oncogeneexpressing mice) are used to induce tumors in mice. Mice are treatedwith nuclear pore complex (NPC) assembly and disassembly regulatorsincluding but not limited to small molecules, siRNAs, shRNAs, microRNAs,mRNAs, gRNA/CRISPR and the development of cancer is detected byanalyzing is tumor size (or by blood analyses for blood malignanciessuch as leukemias and lymphomas). Alternatively, cancer cells expressinga luciferase or another bioluminescent reporter or fluorescent reporterare used for injections and tumor development is followed by in vivooptical imaging such as Xenogen, IVIS® Spectrum, NightOWL LB 983, CRiMaestro 2, or other animal imaging systems. After treatment, tumors arecollected, weighted, and the number of NPCs is analyzed in isolatedtissues, cells and nuclei from the tumors by microscopy analysis usingantibodies against nuclear pore complex component or by measuring theprotein or RNA levels of NPC proteins in tumors or cell extracts.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of treating a proliferative disease ordisorder in an individual in need thereof, comprising: administering tothe individual a therapeutically effective amount of an agent thatinhibits nuclear pore complex assembly or induces nuclear pore complexdisassembly, wherein the nuclear pore complex assembly is not inhibitedin non-dividing cells or the nuclear pore complex disassembly is notinduced in the non-dividing cells, wherein the proliferative disease ordisorder is cancer, wherein the cancer has a mutation in Ras gene, andwherein the agent is a small interfering RNA (siRNA) or short hairpinRNA (shRNA) that targets Nup160 of the nuclear pore complex.
 2. Themethod of claim 1, wherein the agent inhibits expression of a componentof the nuclear pore complex.
 3. The method of claim 1, wherein the agentpromotes degradation of a component of the nuclear pore complex.
 4. Themethod of claim 1, further comprising administering an additionaltherapeutic agent.
 5. The method of claim 1, wherein the cancer ismelanoma cancer.
 6. The method of claim 1, wherein the mutation is aG12D mutation.
 7. The method of claim 1, wherein the administering tothe individual the therapeutically effective amount of the agentcomprises contacting a cell having a number of nuclear pore complexeswith the agent wherein the agent reduces the number of the nuclear porecomplexes in the cell.
 8. The method of claim 1, wherein the cancer iscolorectal cancer.